Boronic compound complexing reagents and highly stable complexes

ABSTRACT

Boron compound completing reagents, intermediate reagents of those reagents and methods of synthesizing these reagents. These reagents, including those shown as General Formula CII may be used, after further reactions described herein, to complex with boronic compounds, such as phenylboronic acid or derivatives thereof. ##STR1## In one embodiment, group Z comprises a spacer selected from a saturated or unsaturated chain up to about 0 to 6 carbon equivalents in length, an unbranched saturated or unsaturated chain of from about 6 to 18 carbon equivalents in length with at least one of intermediate amide and disulfide moieties, and a polyethylene glycol chain of from 3 to 12 carbon equivalents in length; group R 2  is one of an alkyl and a methylene with an electronegative moiety; and BAS is a biologically active species.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 08/691,930, filed Aug. 5,1996, now U.S. Pat. No. 5,777,148 which is a continuation in part ofU.S. application Ser. No. 08/188,531 which was filed on Jan. 28, 1994and is now U.S. Pat. No. 5,594,191 and which is hereby incorporatedherein by reference.

This application also follows application Ser. No. 08/188,460, nowabandoned; Ser. No. 08/188,958, now U.S. Pat. No. 5,594,111 and Ser. No.08/188,176, now U.S. Pat. No. 5,623,055, each of which is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of bioconjugate preparation,and more particularly, to a class of boronic compound complexingreagents useful for the conjugation of biological macromolecules, andthe method of making and using such reagents.

BACKGROUND OF THE INVENTION

Bioconjugation is a descriptive term for the joining of two or moredifferent molecular species by chemical or biological means, in which atleast one of the molecular species is a biological macromolecule. Thisincludes, but is not limited to, conjugation of proteins, peptides,polysaccharides, hormones, nucleic acids, liposomes and cells, with eachother or with any other molecular species that add useful properties,including, but not limited to, drugs, radionuclides, toxins, haptens,inhibitors, chromophores, fluorophores, ligands, etc. Immobilization ofbiological macromolecules is also considered a special case ofbioconjugation in which the macromolecule is conjugated, eitherreversibly or irreversibly, to an insoluble support. Bioconjugation isutilized extensively in biochemical, immunochemical and molecularbiological research. Major applications of bioconjugation include;detection of gene probes, enzyme-linked immuno solid-phase assay,monoclonal antibody drug targeting and medical imaging.

Bioconjugates are generally classified as either direct or indirectconjugates. Direct conjugates encompass those in which two or morecomponents are joined by direct covalent chemical linkages.Alternatively, indirect conjugates encompass those in which two or morecomponents are joined via an intermediary complex involving a biologicalmacromolecule. The system described herein is the first to enable theformation of indirect conjugates without dependence upon an intermediarybiological macromolecule.

AVIDIN-BIOTIN SYSTEM

Although numerous methods of indirect bioconjugate preparation have beendescribed, a significant number of those reported in the literature havebeen prepared by exploiting the Avidin-Biotin system, in which, thebinding specificity of the protein Avidin (purified from egg white), orStreptavidin (purified from the bacterium Streptomyces avidinii), towardthe cofactor Biotin (vitamin H) is utilized to bridge an Avidinconjugated macromolecule with a biotinylated macromolecule. Both Avidinand Streptavidin possess four Biotin binding sites of very high affinity(K=10¹⁵ mol⁻¹).

The Avidin-Biotin system has been utilized extensively for enzyme-linkedimmuno solid-phase assay (ELISA), in which an enzyme-Avidin conjugate(useful for detection by reaction with the enzyme's substrate to afforda colored or chemiluminescent product) is employed to detect thepresence of a biotinylated antibody, after first binding the antibody toan immobilized antigen or hapten. Applications of the Avidin-Biotinsystem number in the hundreds, and have recently been reviewed (Wilchek,M. and Bayer, E. A., (1990) Methods in Enzymology, 184). Althoughutilized extensively, several limitations are known to be associatedwith the Avidin-Biotin system, which include nonspecific bindinggenerally attributed to the basicity of the Avidin molecule, nonspecificbinding attributed to the presence of carbohydrate residues on theAvidin molecule, and background interference associated with thepresence of endogenous Biotin, which is ubiquitous in both eukaryoticand prokaryotic cells.

DIGOXIGENIN α-DIGOXIGENIN SYSTEM

An alternative indirect bioconjugation system designed to overcome someof the limitations associated with the Avidin-Biotin system has recentlybeen developed for the detection of gene probes by ELISA (Kessler, C.,Holtke, H.-J., Seibl, R., Burg, J. and Muhlegger, K., (1990) Biol. Chem.Hoppe-Seyler, 371, 917-965. This system involves the use of the steroidhapten Digoxigenin, an alkaloid occuring exclusively in Digitalisplants, and Fab fragments derived from polyclonal sheep antibodiesagainst Digoxigenin (α-Digoxigenin). The high specificity of the variousα-Digoxigenin antibodies affords low backgrounds and eliminates thenon-specific binding observed in Avidin-Biotin systems.Digoxigenin-labeled DNA and RNA probes can detect single-copy sequencesin human genomic Southern blots. The development of the Digoxigeninα-Digoxigenin system has recently been reviewed (Kessler, C. (1990) inAdvances in Mutagenesis Research (Obe, G. ed.) pp. 105-152,Springer-Verlag, Berlin/Heidelberg). The Digoxigenin α-Digoxigeninsystem is the most recent representative of several hapten-antibodysystems now utilized for bioconjugation.

IMMOBILIZED PHENYLBORONATES

Phenylboronic acids are known to interact with a wide range of polarmolecules having certain requisite functionalities. Complexes of varyingstability, involving 1,2 diols, 1,3-diols, 1,2-hydroxy acids,1,3-hydroxy acids, 1,2-hydroxylamines, 1,3-hydroxylamines, 1,2-diketonesand 1,3-diketones, are known to form with either neutral phenylboronicacid or phenylboronate anion. Consequently, immobilized phenylboronicacids have been exploited as chromatographic supports to selectivelyretain, from diverse biological samples, those molecular species havingthe requisite functionalities. Many important biological moleculesincluding carbohydrates, catecholamines, prostaglandins,ribonucleosides, and steroids contain the requisite functionalities, andhave been either analyzed or purified in this manner. The use ofphenylboronic acid chromatographic media for the isolation andseparation of biological molecules has been discussed in several reviews(Singhal, R. P. and DeSilva, S. S. M. (1992) Adv. Chromatog., 31,293-335; Mazzeo, J. R. and Krull, I. S. (1989) BioChromatog., 4,124-130; and Bergold, A. and Scouten, W. H. (1983) in Solid PhaseBiochemistry (Scouten, W. H. ed.) pp. 149-187, John Wiley & Sons, NewYork).

Phenylboronic acid, like boric acid, is a Lewis acid, and ionizes not bydirect deprotonation, but by hydration to give the tetrahedralphenylboronate anion (pK_(a) =8.86). Phenylboronic acid is three timesas strong an acid as boric acid. Ionization of phenylboronic acid is animportant factor in complex formation, in that, upon ionization, boronchanges from trigonal coordination (having average bond angles of 120°and average bond lengths of 1.37≈) to the tetrahedrally coordinatedanion (having average bond angles of 109° and average bond lengths of1.48≈).

Molecular species having cis or coaxial 1,2-diol and 1,3-diolfunctionalities, and particularly carbohydrates, are known to complexwith immobilized phenylboronate anion, to form cyclic esters underalkaline aqueous conditions (Lorand, J. P. and Edwards, J. O. (1959) J.Org. Chem., 24, 769).

Acidification of 1,2-diol and 1,3-diol complexes to neutral pH is knowto release the diol containing species, presumably due to hydrolysis ofthe cyclic ester. Coplanar aromatic 1,3-diols, like1,8-dihydroxynaphthalene, are known to complex even under acidicconditions due to the hydrolytic stability of six-membered cyclicboronic acid esters (Sienkiewicz, P. A. and Roberts, D. C. (1980) J.Inorg. Nucl. Chem., 42, 1559-1571). Molecular species having pendant1,2-hydroxylamine, 1,3-hydroxylamine, 1,2-hydroxyamide,1,3-hydroxyamide, 1,2-hydroxy-oxime and 1,3-hydroxyoxime functionalitiesare also known to reversibly complex with phenylboronic acid underalkaline aqueous conditions similar to those associated with theretention of diol containing species (Tanner, D. W. and Bruice, T. C.(1967) J. Amer. Chem. Soc., 89, 6954).

PHENYLBORONATE BIOCONJUGATES

Ortho-substituted acetamidophenylboronic acids have been proposed aspotential linkers for selective bioconjugation via the vicinal diolmoieties of the carbohydrate residues associated with glycoproteins(Cai, S. X. and Keana, J. F. W. (1991) Bioconjugate Chem., 2, 317-322).

Phenylboronic acid bioconjugates derived from3-isothiocyanatophenylboronic acid have been successfully utilized forappending radioactive technetium dioxime complexes to monoclonalantibodies for use in medical imaging (Linder, K. E., Wen, M. D.,Nowotnik, D. P., Malley, M. F., Gougoutas, J. Z., Nunn, A. D. andEckelman, W. C. (1991) Bioconjugate Chem., 2, 160-170; Linder, K. E.,Wen, M. D., Nowotnik, D. P., Ramalingam, K., Sharkey, R. M., Yost, F.,Narra, R. K. and Eckelman, W. C. (1991) Bioconjugate Chem, 2, 407-414).

3-Aminophenylboronic acid has been covalently appended to proteins by avariety of chemical methods and the resulting phenylboronic acidbioconjugates tested for their binding of D-sorbitol, D-mannose andglycated hemoglobin (GHb). The interactions proved to be reversible andof very low affinity rendering the bioconjugates of very limitedpractical use. Similarly, an alkaline phosphatase phenylboronic acidbioconjugate used in an attempted enzyme-linked assay for the detectionof GHb failed to detect the presence of glycated protein (Frantzen, F.,Grimsrud, K., Heggli, D. and Sundrehagen, E. (1995) Journal ofChromatography B, 670, 37-45).

Although immobilized phenylboronates have been utilized forchromatographic separation of biological molecules having the requisitefunctionalities, notwithstanding the substantial amount of research intobioconjugation, and the substantial amount of investment in this field,the selectivity of phenylboronic acid has not heretofore beensuccessfully exploited to enable the conjugation of biologicalmacromolecules with one another or with other molecular species that adduseful properties.

SUMMARY OF THE INVENTION

The present invention relates to a novel class of boron compoundcomplexing reagents useful for the preparation of bioconjugates, and themethod of making and using such reagents. In one embodiment, the boroncompound is phenylboronic acid, or derivatives thereof, which complexwith the complexing reagents of the present invention. Unless otherwisenoted, the phrase phenylboronic acid complexing reagent is used hereinto include the broader class of boron compound complexing reagents, andthe phrase phenylboronic acid is used herein to include the broaderclass of boron compounds which complex with the boron compoundcomplexing reagents. In the present invention, in the place of prior artAvidin-Biotin and Digoxigenin α-Digoxigenin systems, boron compoundcomplexing reagents are utilized in conjunction with the boron compound,such as phenylboronic acid reagents (many of which are known in theprior art) to facilitate chemical conjugation without the use ofintermediary biological macromolecules. Bioconjugate preparation ofteninvolves the conjugation of several components including, but notlimited to, proteins, peptides, polysaccharides, hormones, nucleicacids, liposomes and cells, with each other or with any other molecularspecies that add useful properties, including, but not limited to,drugs, radionuclides, toxins, haptens, inhibitors, fluorophores,ligands, solid-phase supports, and boron compound complexing reagentsconjugates. These various components utilized in bioconjugatepreparation will collectively and individually be termed biologicallyactive species or bioactive species.

Reagents suitable for the modification of a bioactive species for thepurpose of incorporating a phenylboronic acid complexing moiety forsubsequent conjugation to a different (or the same) bioactive specieshaving pendant phenylboronic acid moieties are of the general formula ofGeneral Formula CI: ##STR2## Group R₁ is a reactive electrophilic ornucleophilic moiety suitable for reaction of the putative phenylboronicacid complexing reagent with a bioactive species. Group Z is a spacerselected from a saturated or unsaturated chain of from about 0 to 6carbon equivalents in length, an unbranched saturated or unsaturatedchain of from about 6 to 18 carbon equivalents in length with at leastone of intermediate amide or disulfide moieties, and a polyethyleneglycol chain of from about 3 to 12 carbon equivalents in length. GroupR₂ is selected from an alkyl (e.g., methyl, ethyl, etc.) and a methylenebearing an electronegative substituent.

Reaction of a reagent of General Formula CI with a bioactive speciesaffords a conjugate having pendant putative phenylboronic acidcomplexing moieties (one or more) of General Formula CII, ##STR3## Thesymbol labeled BAS represents a biologically active species (orbioactive species) that may or may not contain a portion of a reactivemoiety (which may itself have a spacer) used to attach the bioactivespecies to the reagent. It will be appreciated that, in manyembodiments, several identical reagents of the general formula ofGeneral Formula CI will react with a single BAS molecule. For example,if the BAS is a protein, many phenylboronic acid complexing reagentswill react with the protein, each reacting at one of the several siteson the protein which are reactive with the R₁ group. Group Z in GeneralFormula CII is a spacer selected from a saturated or unsaturated chainof from about 0 to 6 carbon equivalents in length, an unbranchedsaturated or unsaturated chain of from about 6 to 18 carbon equivalentsin length with at least one of intermediate amide or disulfide moieties,and a polyethylene glycol chain of from about 3 to 12 carbon equivalentsin length. Group R₂ is selected from an alkyl (e.g., methyl, ethyl,etc.) and a methylene bearing an electronegative substituent.

The reagent of General Formula CII may be further reacted to afford aclass of conjugate of boronic compound complexing reagents, e.g.,reagents with one or more pendant phenylboronic acid complexing moietiesof General Formula CIV: ##STR4##

With respect to the conjugate of General Formula CIV, group Z is aspacer selected from a saturated or unsaturated chain of from about 0 to6 carbon equivalents in length, an unbranched saturated or unsaturatedchain of from about 6 to 18 carbon equivalents in length with at leastone of intermediate amide or disulfide moieties, and a polyethyleneglycol chain of from about 3 to 12 carbon equivalents in length. GroupR₃ is selected from one of an H, an alkyl, and a methylene or ethylenewith an electronegative substituent. Finally, the symbol BAS representsthe bioactive species that may or may not contain a portion of areactive moiety used to attach the bioactive species.

The compositions of General Formula CII and General Formula CIV, andmethods for their preparation are described herein and are the subjectof my copending application, titled Boronic Compound Complexing Reagentsand Highly Stable Complexes, U.S. Ser. No. 08/691,930, now U.S. Pat. No.5,777,148 and filed Aug. 5, 1996 by Mark L. Stolowitz, Robert J. Kaiser,and Kevin P. Lund which copending application is hereby incorporatedherein by reference.

Phenylboronic acid reagents, many of which are known in the prior art,as well as those described in greater detail in my copendingapplication, titled Plenylboronic Acid Complexes for BioconjugatePreporation, and filed on Jan. 28 1994, U.S. Pat. No. 5,677,431, whichis incorporated herein by reference, may be appended to a biologicallyactive species to afford a conjugate having pendant phenylboronic acidmoieties (one or more) of General Formula CV: ##STR5## wherein thesymbol labeled BAS* represents a second bioactive species, that mayinclude a linker portion and that may differ from the bioactive specieslabeled BAS. The BAS* may also include a portion of a reactive moietyused to attach the bioactive species to the phenylboronic acid reagent.

A conjugate of General Formula CIV, with at least one biologicallyactive species and having pendent phenylboronic acid complexing moieties(one or more), may be complexed with a conjugate of General Formula CV,prepared from a second bioactive species BAS* and having pendantphenylboronic acid moieties (one or more), to afford a bioconjugate ofGeneral Formula CVI, ##STR6## wherein the symbols labeled BAS and BAS*,and groups Z and R₃ are as were previously defmed. In this manner,biological macromolecules may be conjugated to one another or with otherfunctionalities which impart useful properties.

Bioconjugates of General Formula CVI may be prepared in buffered aqueoussolution or organic solvents. The bioconjugate is formed within a fewminutes over a range of temperatures of from about 4° C. to 70° C. Thestability of the bioconjugate in aqueous solution at a given pH andtemperature is significantly influenced by substituent group R₃.Bioconjugates of General Formula CVI, are stable in aqueous solutions ofapproximate pH greater than 3.5 and less than 10.5.

The bioconjugation reaction (phenylboronic acid complexation) isinsensitive to significant variations in ionic strength, the presence oforganic solvents, the presence of detergents, and the presence ofchaotropic agents (protein denaturants), which are incompatible withprior art indirect labeling systems wherein the structure of abiological macromolecule must be maintained to preserve requisitebinding properties. In most instances, the constraints governing theformation of bioconjugates, by the system herein described, are limitedto those imposed by the conditions required to maintain viability of thebioactive species.

In summary, boron compound complexing reagents, intermediate reagents ofthose reagents and methods of synthesizing the reagents are described.These reagents, including those shown as General Formulas CI and CII maybe used, after further reactions described herein, to complex withboronic compounds, such as phenylboronic acid or derivatives thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the utilization of putative phenylboronic acidcomplexing reagents of General Formula I and phenylboronic acidcomplexing reagents of General Formula III, to prepare conjugates ofGeneral Formula IV which may, in turn, be utilized to preparebioconjugates of General Formula VI.

FIG. 2 summarizes the preparation of alkyl 4- and5-aminomethylsalicylates, synthetic intermediates leading to reagents ofGeneral Formula I, wherein R₂ is an alkyl group, e.g., methyl, ethyl,propyl, etc. Alkyl 4- and 5-aminomethylsalicylates are also usefulsynthetic intermediates leading to reagents of General Formula III.

FIG. 3 summarizes the preparation of alkyl 4- and5-aminomethylsalicylates, synthetic intermediates leading to reagents ofGeneral Formula I, wherein R₂ is a methylene group bearing anelectronegative moiety, e.g., carboxymethyl, cyanomethyl, methoxymethyl,etc.

FIG. 4 summarizes the preparation of 4- and5-aminomethylsalicylhydroxamic acids, synthetic intermediates leading toreagents of General Formula III, wherein R₃ is one of either an alkylgroup or a methylene or ethylene group bearing an electronegativemoiety.

FIG. 5 summarizes the synthesis of reagents of General Formulas I andIII, wherein R₂ is one of either an alkyl group or a methylene groupbearing an electronegative moiety, and wherein R₁ is selected fromeither imidazolide, hydrazide and N-hydroxysuccinimidyl ester moieties.

FIG. 6 summarizes the synthesis of reagents of General Formulas I andIII, wherein R₂ is one of either an alkyl group or a methylene groupbearing an electronegative moiety, and wherein R₁ is selected fromeither bromo, chloro, iodo, maleimide, dithiopyridyl and imidate estermoieties.

FIG. 7 summarizes the synthesis of reagents of General Formulas I andIII, wherein R₁ is an N-hydroxysuccinimidy ester, and wherein Z anunbranched saturated or unsaturated chain of from about 6 to 18 carbonequivalents in length with at least one of either an intermediate amideor disulfide moiety.

FIG. 1' (labeled in the Figures as FIG. 8 but referred to as FIG. 1' inthe text) illustrates the utilization of putative phenylboronic acidcomplexing reagents of General Formula CI and phenylboronic acidcomplexing reagents of General Formula CIII, to prepare conjugates ofGeneral Formula CIV which may, in turn, be utilized to preparebioconjugates of General Formula CVI.

FIG. 2' (labeled in the Figures as FIG. 9 but referred to as FIG. 2' inthe text) summarizes the preparation of alkyl 4- and5-aminomethyl-2,6-dihydroxybenzoates, synthetic intermediates leading toreagents of General Formula CI, wherein R₂ is an alkyl group, e.g.,methyl, ethyl, propyl, etc. Alkyl 4- and5-aminomethyl-2,6-dihydroxybenzoates are also useful syntheticintermediates leading to reagents of General Formula CIII.

FIG. 3' (labeled in the Figures as FIG. 10 but referred to as FIG. 3' inthe text) summarizes the preparation of alkyl 4- and5-aminomethyl-2,6-dihydroxybenzoates, synthetic intermediates leading toreagents of General Formula CI, wherein R₂ is a methylene group bearingan electronegative moiety, e.g., carboxymethyl, cyanomethyl,methoxymethyl, etc.

FIG. 4' (labeled in the Figures as FIG. 11 but referred to as FIG. 4' inthe text) summarizes the preparation of 4- and5-aminomethyl-2,6-dihydroxybenzohydroxamic acids, syntheticintermediates leading to reagents of General Formula CIII, wherein R₃ isone of either an alkyl group or a methylene or ethylene group bearing anelectronegative moiety.

FIG. 5' (labeled in the Figures as FIG. 12 but referred to as FIG. 5' inthe text) summarizes the synthesis of reagents of General Formulas CIand CIII, wherein R₂ is one of either an alkyl group or a methylenegroup bearing an electronegative moiety, and wherein R₁ is selected fromeither imidazolide, hydrazide and N-hydroxysuccinimidyl ester moieties.

FIG. 6' (labeled in the Figures as FIG. 13 but referred to as FIG. 6' inthe text) summarizes the synthesis of reagents of General Formulas CIand CIII, wherein R₂ is one of either an alkyl group or a methylenegroup bearing an electronegative moiety, and wherein R₁ is selected fromeither bromo, chloro, iodo, maleimide, dithiopyridyl and imidate estermoieties.

FIG. 7' (labeled in the Figures as FIG. 14 but referred to as FIG. 7' inthe text) summarizes the synthesis of reagents of General Formulas CIand CIII, wherein R₁ is an N-hydroxysuccinimidy ester, and wherein Z anunbranched saturated or unsaturated chain of from about 6 to 18 carbonequivalents in length with at least one of either an intermediate amideor disulfide moiety.

DETAILED DESCRIPTION OF THE INVENTION PHENYLBORONIC ACID COMPLEXINGREAGENTS AND CONJUGATES DERIVED FROM 4 OR 5-AMINOMETHYSALICYLIC ACID

This section relates to a class of phenylboronic acid complexingreagents and conjugates derived from 4- or 5-aminomethylsalicylic acidand refers to FIGS. 1 through 7. Reagents and conjugates derived from 4-or 5-aminomethylsalicylic acid are the subject of my copendingapplications titled: Boronic Compound Complexing Reagents and Complexes,Filed Aug. 5, 1996 and titled: Boronic Compound Complexing Reagents andComplexes, Filed Aug. 5, 1996 and Ser. No. 08/689,341 and U.S. Pat. No.5,744,627, respectively. The discussion of the preparation of thesecompounds herein is to facilitate the overall understanding of thedistinct and novel invention described herein. By presenting thepreparation of various compounds from a similar family of complexingreagents and conjugates, it is intended that the fill scope of theclaimed invention is recognized.

A three-step process which utilizes reagents of General Formula I forthe preparation of bioconjugates is summarized in FIG. 1. Initially, areagent of General Formula I is selected that is comprised of anappropriate reactive electrophilic or nucleophic group R₁ suitable forreaction with the desired biologically active species. ##STR7## Group R₁is a reactive electrophilic or nucleophilic moiety suitable for reactionof the putative phenylboronic acid complexing reagent with a bioactivespecies. Group R₁ is preferably selected from, but not limited to,acrylamide, bromo, dithiopyridyl, bromoacetamide, hydrazide,N-hydroxysuccinimidyl ester, N-hydroxysulfosuccinimidyl ester, imidateester, imidazolide, iodo, iodoacetamide, maleimide, amino and thiolmoieties.

Group Z is a spacer selected from a saturated or unsaturated, preferablyunbranched, chain of from about 0 to 6 carbon equivalents in length, anunbranched saturated or unsaturated chain of from about 6 to 18 carbonequivalents in length with at least one of intermediate amide ordisulfide moieties, and a polyethylene glycol chain of from about 3 to12 carbon equivalents in length. Group Z is preferably selected from anunbranched alkyl chain of general formula (CH₂)_(n), wherein n=1 to 6.

Group R₂ is selected from an alkyl (e.g., methyl, ethyl, etc.) and amethylene bearing an electronegative substituent. An electronegativesubstituent is a substituent with a negative dipole moment, e.g., CN,COOH, etc. Group R₂ is preferably selected from one of CH₃, CH₂ CH₃, CH₂CN, CH₂ COOH, CH₂ CONH₂ and CH₂ OCH₃.

The next step in a three-step process in the preparation ofbioconjugates is to condense the appropriate reagent with the bioactivespecies to yield a conjugate of General Formula II: ##STR8## In GeneralFormula II, Z and R₂ are as defined above, and BAS represents abiologically active species which may or may not contain a portion of areactive moiety (and any spacer) used to attach the biologically activespecies to the reagent.

Next, the conjugate is reacted with a hydroxylamine derivative of thegeneral formula NH₂ OR₃, wherein R₃ is selected from either an H, analkyl (e.g., methyl, ethyl, etc.), or a methylene or ethylene with anelectronegative moiety. Suitable hydroxylamine derivatives include, butare not limited to, NH₂ OH, NH₂ OCH₃, NH₂ OCH₂ CN, NH₂ OCH₂ COOH, NH₂OCH₂ CONH₂ and NH₂ OCH₂ CH₂ OH. The resulting conjugate has the generalformula of General Formula IV: ##STR9## In General Formula IV, Groups Zand BAS are as defmed above, and R₃ is selected from one of an H, analkyl, and a methylene moiety with an electronegative substituent.

The conjugate of General Formula IV is then complexed with aphenylboronic acid having the general formula of General Formula V:##STR10## wherein the symbol labeled BAS* represents a secondbiologically active species, that may include a linker portion anddiffer from the biologically active species labeled BAS of thecomplexing reagent. The BAS* may also include a portion of a reactivemoiety used to attach the bioactive species to the phenylboronic acidreagent. The complexation yields the stereoisomeric complex (abouttetrahedral boron) of General Formula VI: ##STR11##

SYNTHESIS OF PUTATIVE PHENYLBORONIC ACID COMPLEXING REAGENTS OF GENERALFORMULA I ##STR12##

Reagents of General Formula I are derived from either 4- or5-methylsalicylic acid. In each instance, the reagent is ultimatelyprepared from a synthetic intermediate which is either an alkyl 4- or5-aminomethylbenzoate. FIG. 2 summarizes the preparation of alkyl 4- and5-aminomethylbenzoates, synthetic intermediates leading to reagents ofGeneral Formula I, wherein R₂ is an alkyl group, e.g., methyl, ethyl,etc. FIG. 2 shows an example where R₂ is a methyl group. Initially, instep 1, either 4- or 5-methyl-salicylic acid is esterified to afford thecorresponding alkyl 4- or 5-methylsalicylate. In step 2, the ester isbrominated with N-bromosuccinimide and benzoyl peroxide catalyst toafford the corresponding benzyl bromide. In step 3, the benzyl bromideis alkylated with sodium azide to afford the corresponding benzyl azide.Finally, in step 4, the benzyl azide is subjected to palladium catalyzedhydrogenation in the presence of HCl to afford the corresponding benzylamine hydrochloride.

FIG. 3 summarizes the preparation of synthetic intermediates leading toreagents of General Formula I, wherein group R₂ is a methylene bearingan electronegative moiety, for example, carboxymethyl, acetamidomethyland cyanomethyl 4- or 5-aminomethylsalicylate. It is to be appreciatedthat other contemplated substituents for group R₂ may be prepared by asimilar synthesis. Initially, in step 1 of FIG. 3, the alkyl 4- or5-aminomethylsalicylate, prepared as summarized in FIG. 2, is reactedwith di-tert-butyl dicarbonate and triethylamine in methanol to affordthe corresponding protected methylN-(tert-butoxycarbonyl)aminomethylbenzoate. In step 2, the methyl esteris cleaved by reaction with potassium trimethylsilanolate and worked upin aqueous acid to afford the corresponding benzoic acid. In step 3, thebenzoic acid is alkylated by reaction with either an α-haloacid,α-haloacetamide or α-haloacetonitrile and triethylamine to afford thecorresponding carboxymethyl, acetamidomethyl or cyanomethyl ester,respectively. Finally, in step 4, the N-(tert-butoxycarbonyl) protectinggroup is removed by reaction with anhydrous HCl in tetrahydrofuran toafford the corresponding benzyl amine hydrochloride.

Another reagent which is the acrylic acid amide ofaminomethyl-salicylate, can be prepared in a single step, using the endproduct of FIG. 3 by condensing acrylic acid anhydride or acryloylchloride with aminomethyl-salicylate.

FIG. 5 summarizes the synthesis of reagents of General Formula I,wherein group R₂ is one of either an alkyl or a methylene bearing anelectronegative moiety, and wherein group R₁ is selected from eitherimidazolide, hydrazide and N-hydroxysuccinimidyl ester moieties. Thesereagents are each prepared by a two-step process in which an aliphaticacid anhydride is utilized in the first step. Initially, an alkyl 4- or5-aminomethylsalicylate, prepared as summarized in either FIG. 2 or FIG.3, is condensed of an aliphatic acid anhydride preferably selected from,but not limited to, either succinic anhydride, glutaric anhydride,maleic anhydride and glycolic acid anhydride, in an aprotic organicsolvent, which results in the introduction of a spacer (group Z) havinga free terminal carboxylic acid moiety. In the case where the aliphaticacid anhydride is maleic anhydride in this condensation reaction, theresulting Z group is unsaturated as it contains an alkene group.Subsequently, the carboxylic acid moiety is further functionalized byreaction with either N,N-carbonyldiimidazole, isobutylchloroformate andtert-butyl carbazate, or N,N-dicyclohexylcarbodiimide andN-hydroxysuccinimide to afford the corresponding imidazolide, protectedhydrazide and N-hydroxysuccinimidyl ester, respectively. In the instanceof the protected hydrazide, the N-(tert-butoxycarbonyl) protecting groupis removed by contacting the reagent with anhydrous hydrochloric acid.

FIG. 6 summarizes the synthesis of reagents of General Formula I,wherein group R₂ is one of either an alkyl or a methylene bearing anelectronegative moiety, and wherein group R₁ is selected from eitheracrylamide, bromoacetamide, chloroacetamide, maleimide, dithiopyridyland imidate ester moieties. Reagents of General Formula I, wherein groupR₁ is selected from either bromo and chloro moieties, are prepared bycondensing an alkyl 4- or 5-aminomethylsalicylate, prepared as summarizein either FIG. 2 or FIG. 3, with either bromoacetic acid anhydride orchloroacetic acid, respectively. The homologous iodo reagent is preparedby halogen exchange of the chloro reagent with sodium iodide. Reagentsof General Formula I, wherein R₁ is selected from either bromo, chloro,iodo, bromoacetamide, chloroacetamide and iodoacetamide moieties, maynot be conveniently prepared when R₃ is H, due to the potential forintermolecular alkylation of the unprotected hydroxamate. Reagents ofGeneral Formula I, wherein group R₁ is selected from either maleimide ordithiopyridyl moieties, are prepared by condensing an alkyl 4- or5-aminomethylsalicylate, prepared as summarized in either FIG. 2 or FIG.3, with an N-hydroxysuccinimidyl ester of an aliphatic carboxylic esterwhich bears either a terminal maleimide to dithiopyridyl moiety. Areagent of General Formula I, wherein R₁ is an imidate ester moiety, isprepared by a two-step process in which an alkyl 4- or5-aminomethyl-salicylate, prepared as summarized in either FIG. 2 orFIG. 3, is first condensed with an N-hydroxysuccinimidyl ester of analiphatic carboxylic ester which bears a terminal nitrile moiety.Subsequently, the nitrile moiety is converted to the methyl imidateester by reaction with anhydrous hydrogen chloride in methanol at 0° C.

Reagents of General Formula I, wherein group R₁ is selected from eitherN-hydroxysuccinimidyl ester and dithiopyridyl moieties, may be utilizedas synthetic intermediates to prepare reagents of General Formula I,wherein group Z is an unbranched saturated or unsaturated chain with atleast one of intermediate amide or disulfide moieties.

Reagents of General Formula I, wherein group R₁ is anN-hydroxysuccinimidyl ester moiety, may be condensed with compoundshaving primary aliphatic amine moieties of the general formula R₁--Z--NH₂, wherein Z₂ is an unbranched saturated or unsaturated chainpreferably of from about 1 to 5 carbon equivalents in length, to affordreagents of General Formula I, wherein group Z is an unbranchedsaturated or unsaturated chain with at least one of intermediate amidemoieties.

Alternatively, N-hydroxysuccinimidyl ester reagents of General FormulaI, preferably derived from a dicarboxylic acid selected from eithersuccinic acid, maleic acid, fumaric acid, acetylenedicarboxylic acid andglutaric acid, may be condensed with compounds having primary aliphaticamine moieties of the general formula HO₂ C--Z₂ --NH₂, wherein Z₂ is anunbranched saturated or unsaturated chain preferably of from about 1 to5 carbon equivalents in length, preferably selected from, but notlimited to, either glycine, β-alanine, aminopropiolic acid,4-aminobutyric acid and 6-aminocaproic acid, to afford compounds havingfree terminal carboxylic acid moieties which may be furtherfunctionalized in accordance with FIG. 5 to afford reagents of GeneralFormula I, wherein Z is an unbranched saturated or unsaturated chainwith at least one of intermediate amide moieties. This process issummarized in FIG. 7 for the synthesis of a reagent of General FormulaI, wherein R₁ is an N-hydroxysuccinimidyl ester, and wherein Z is anunbranched saturated or unsaturated chain with at least one of anintermediate amide moiety.

Reagents of General Formula I, wherein group R₁ is a dithiopyridylmoiety, may be condensed with compounds having terminal thiol moietiesof the general formula R₁ --Z₂ --SH, wherein Z₂ is an unbranchedsaturated or unsaturated chain preferably of from about 1 to 5 carbonequivalents in length, to afford reagents of General Formula I, whereingroup Z is an unbranched saturated or unsaturated chain with at leastone of intermediate disulfide moieties.

Alternatively, dithiopyrinyl reagents of General Formula I, preferablyderived from a mercaptocarboxylic acid selected from eithermercaptoacetic acid, β-mercaptopropionic acid, mercaptopropiolic acid,4-mercaptobutyric acid and 6-mercaptocaproic acid, may be condensed withcompounds having a thiol moiety of the general formula HO₂ C--Z₂ --SH,wherein Z₂ is an unbranched saturated or unsaturated chain preferably offrom about 1 to 5 carbon equivalents in length, preferably selectedfrom, either mercaptoacetic acid, β-mercaptopropionic acid,mercaptopropiolic acid, 4-mercaptobutyric acid and 6-mercaptocaproicacid, to afford compounds having free terminal carboxylic acid moietieswhich may be further functionalized in accordance with FIG. 5 to affordreagents of General Formula I, wherein group Z is an unbranchedsaturated or unsaturated chain with at least one of intermediatedisulfide moieties. This process is summarized in FIG. 7 for thesynthesis of a reagent of General Formula I, wherein R₁ is anN-hydroxysuccinimidyl ester, and wherein Z is an unbranched saturated orunsaturated chain with at least one of an intermediate disulfide moiety.

Reagents of General Formula I, wherein group Z is a polyethylene glycolchain of from about 3 to 12 carbon equivalents in length, are preparedby condensing an alkyl 4- or 5-aminomethylsalicylate, prepared assummarized in FIGS. 2 or 3, with a polyethylene glycol reagent havingboth an N-hydroxysuccinimidyl ester moiety and either a reactiveelectrophilic or nucleophilic moiety (or a protected precursor thereof),many of which are commercially available, to afford reagents of GeneralFormula I, wherein group Z is a polyethylene glycol chain of from about3 to 12 carbon equivalents in length.

SYNTHESIS OF PHENYLBORONIC ACID COMPLEXING REAGENTS OF GENERAL FORMULAIII ##STR13##

FIG. 4 summaries the preparation of 4- and5-aminomethylsalicylhydroxamic acids, synthetic intermediates leading toreagents of General Formula III, wherein group R₃ is one of an alkyl ormethylene bearing an electronegative moiety. Initially, in step 1, analkyl 4- or 5-aminomethylsalicylate, prepared as summarized in FIG. 2,is condensed with N-(benzyloxycarbonyl)oxy succinimide to afford thealkyl N-benzyloxycarbonyl protected 4- or 5-aminomethylsalicylate. Instep 2, the phenolic hydroxyl moiety was condensed with benzyl bromideto afford the further protected benzyl ether intermediate. In step 3,the alkyl ester is selectively cleaved by reaction with LiOH to affordthe corresponding benzoic acid. In step 4, the benzoic acid is activatedby reaction with isobutylchloroformate to form a mixed anhydride whichis subsequently reacted with a hydroxylamine derivative preferablyselected from, but not limited to, either NH₂ OH, NH₂ OCH₃, NH₂ OCH₂ CN,NH₂ OCH₂ COOH, NH₂ OCH₂ CONH₂, NH₂ OCH₂ CH₂ OH and NH₂ OCH₂ OCH₃ toafford the corresponding protected hydroxamic acid. Finally, in step 5,both the amine and phenolic hydroxyl moieties are simultaneouslydeprotected by palladium catalyzed hydrogenation in the presence of HClto afford the corresponding 4- or 5-aminomethylsalicylhydroxamic acidhydrochloride. These synthetic intermediates, which are structurallyrelated to the corresponding alkyl 4- or 5-aminomethylsalicylates, maybe further functionalized in accordance with either FIG. 5 or FIG. 6 toafford reagents of General Formula III.

PREPARATION OF PHENYLBORONIC ACID COMPLEXING CONJUGATES OF GENERALFORMULA IV

At this point, the putative phenylboronic acid complexing reagents ofGeneral Formula I may be reacted with a suitable biologically activespecies to yield the conjugate of the general formula of General FormulaII: ##STR14##

The conjugate of General Formula II is next condensed with ahydroxylamine derivative to yield the corresponding phenylboronic acidcomplexing conjugate of the general formula of General Formula IV:##STR15##

Suitable hydroxylamine derivatives are preferably selected from, but notlimited to, NH₂ OH, NH₂ OCH₃, NH₂ OCH₂ CN, NH₂ OCH₂ COOH, NH₂ OCH₂CONH₂, NH₂ OCH₂ CH₂ OH, NH₂ OCH₂ OCH₃. When group R₂ in General FormulaII is an alkyl group, NH₂ OH is preferably utilized to effect theinterconversion of General Formula II to General Formula IV.

Alternatively, conjugates of General Formula IV may also be prepared bythe route which is described in FIG. 1 which utilizes a reagent ofGeneral Formula III: ##STR16##

Phenylboronic acid complexing reagents of General Formula III may bereacted with a suitable biologically active species to yield theconjugate of the general formula of General Formula IV: ##STR17##

PREPARATION OF BIOCONJUGATES OF GENERAL FORMULA VI

Bioconjugates of General Formula VI may be prepared in buffered aqueoussolutions or organic solvents. Preferred buffers include acetate,citrate, phosphate, carbonate and diglycine. Borate buffers should beavoided due to their ability to complex with the phenylboronic acidcomplexing moiety. Tris, β-hydroxylamine and β-hydroxyacid buffersshould be avoided due to their ability to complex with the phenylboronicacid. The bioconjugate is formed within a few minutes over a range oftemperatures of from about 4° C. to 70° C. The stability of thebioconjugate in aqueous solution at a given pH and temperature isinfluenced, to some extent, by substituent group R₃. Bioconjugates ofGeneral Formula VI are stable in aqueous solutions of approximate pHgreater than 3.5 and less than 10.5. The bioconjugation reaction(phenylboronic acid complexation) is insensitive to significantvariations in ionic strength over the range 0.01 to 1M, the presence oforganic solvents including acetonitrile, methanol, ethanol, isopropanol,butanol, N,N-dimethylformamide and dimethylsulfoxide, the presence ofdetergents, and the presence of chaotropic agents (protein denaturants)including urea, guanidine hydrochloride, guanidine thiocyanate andformamide, which are incompatible with prior art indirect labelingsystems wherein the structure of a biological macromolecule must bemaintained to preserve requisite binding properties. Once formed, thebioconjugates are stable upon removal of water, and can be lyophilizedfor storage.

The stability of the bioconjugate at a given pH is determined to someextent by substituent group R₃. Phenylboronic acid complexes of GeneralFormula VI, wherein group R₃ is H, are stable in buffered aqueoussolutions over the approximate pH range 3.5 to 10.5. Phenylboronic acidcomplexes of General Formula VI, wherein group R₃ is CH₃, are stable inbuffered aqueous solutions over the approximate pH range 4.5 to 10.5.Phenylboronic acid complexes of General Formula VI, wherein group R₃includes an electronegative moiety, are stable in buffered aqueoussolutions over the approximate pH range less than 3.5 to 10.5.

The stability of the phenylboronic acid complex toward acid catalyzedhydrolysis is related to the pK_(a) of the hydroxamic acid participatingin the complex. The lower the pK_(a) of the hydroxamic acid moiety themore stable the complex. Consequently, phenylboronic acid complexes ofGeneral Formula VI wherein group R₃ includes an electronegative moietyexhibit greater stability toward acid catalyzed hydrolysis than do thosein which R₃ is either H or CH₃.

The following examples present a detailed description of the synthesisof reagents of General Formula I and General Formula III.

EXAMPLE I Preparation of Ethyl 4-Aminomethylsylicylate Hydrochloride##STR18## Ethyl 4-Methylsalicylate

4-Methylsalicylic acid (20.0 g, 131 mmoles) was dissolved in ethanol(300 mL) and concentrated sulfuric acid (2.0 mL) was added. The mixturewas refluxed for 40 hours. The volume of the reaction mixture wasreduced to 100 mL, transferred to a separatory funnel, and diluted withchloroform (250 mL) and water (200 mL). Solid sodium bicarbonate wasadded in small portions until the pH of the aqueous layer was about 8(pH test paper). The mixture in the funnel was shaken well and thelayers separated. The organic layer was washed first with water (150 mL)and then saturated aqueous sodium chloride (150 mL). Finally, theorganic solution was dried over anhydrous magnesium sulfate, filtered,and the solvent evaporated to afford 14.0 g (59% yield) of liquid ethyl4-methylsalicylate.

¹ H NMR (300 MHz, CHCl₃ -d) δ1.40 (triplet, J=7 Hz, 3H, CH₂ CH₃), 2.33(singlet, 3H, ArCH₃), 4.38 (quartet, J=7 Hz, 2H, CH₂ CH₃), 6.67(doublet, J=8 Hz, 1H, ArH), 6.78 (singlet, 1H, ArH), 7.72 (doublet, J=8Hz, 1H, ArH), 10.81 (singlet, 1H, OH). ¹³ C NMR (75 MHz, CHCl₃ -d)δ14.0, 21.7, 61.1, 110.1, 117.7, 120.4, 129.7, 146.9, 161.7, 170.3.

Ethyl 4-Bromomethylsalicylic Acid

Ethyl 4-methylsalicylate (13.1 g, 72.5 mmoles) was dissolved in carbontetrachloride (150 mL) and N-bromosuccinimide (13.1 g, 73.2 mmoles) andbenzoyl peroxide (0.2 g, 0.8 mmoles) were added. The mixture wasrefluxed for 3.5 hours and then allowed to cool to room temperature. Thesolids were removed by filtration and the filtrate was evaporated todryness. The crude solid product was crystallized from hexane (100 mL)to afford 5.0 g (27% yield) of ethyl 4-bromomethylsalicylic acid (m.p.64°-66° C.).

¹ H NMR (300 MHz, CHCl₃ -d) δ1.41 (triplet, J=7 Hz, 3H, CH₂ CH₃), 4.40(quartet, J=7 Hz, 2H, CH₂ CH₃), 4.40 (singlet, 2H, CH₂ Br), 6.90(doublet, J=8 Hz, 1H, ArH), 6.99 (singlet, 1H, ArH), 7.82 (doublet, J=8Hz, 1H, ArH), 10.88 (singlet, 1H, OH). ¹³ C NMR (75 MHz, CHCl₃ -d)δ14.0, 31.9, 61.5, 112.4, 117.9, 119.8, 130.5, 145.5, 161.8, 169.9.

Ethyl 4-Aminomethylsalicylate Hydrochloride

Ethyl 4-bromomethylsalicylate (4.8 g, 18.6 mmoles) was dissolved in dryN,N-di-methylformamide (50 mL) and sodium azide (1.2 g, 18.9 mmoles) wasadded. The suspension was stirred at room temperature for 2 hours. Thereaction mixture was then diluted with dichloromethane (150 mL) andextracted with 1N aqueous hydrochloric acid (100 mL), water (100 mL),and saturated aqueous sodium chloride (50 mL). Finally, the solution wasthen dried over anhydrous magnesium sulfate, filtered, and evaporated todryness to give ethyl 4-azidomethylsalicylate as an oil.

Palladium on carbon (0.5 g, 10% w/w!) was added to a 1 L hydrogenationflask under a nitrogen atmosphere. The crude ethyl4-azidomethylsalicylate was dissolved in ethanol (200 mL) andtransferred to the hydrogenation flask. Concentrated aqueoushydrochloric acid (2 mL) was then added, and the flask was affixed tothe Parr hydrogenator. The reaction mixture was shaken under 35 psi ofhydrogen for 4 hours at room temperature. The mixture was then filteredthrough Celite to remove the catalyst, and the filtrate was evaporatedto dryness to afford an off-white solid. Finally, this solid wascrystallized from EtOH to afford 3.1 g (71% yield) of ethyl4-aminomethylsalicylate hydrochloride (m.p. 240°-241° C.).

¹ H NMR (300 MHz, DMSO-d₆) δ1.30 (triplet, J=7 Hz, 3H, CH₂ CH₃), 3.99(singlet, 2H, CH₂ NH₃), 4.33 (quartet, J=7 Hz, 2H, CH₂ CH₃), 7.06(doublet, J=8 Hz, 1H, ArH), 7.15 (singlet, 1H, ArH), 7.77 (doublet, J=8Hz, 1H, ArH), 8.71 (broad singlet, 3H, NH₃), 10.62 (broad singlet, 1H,OH). ¹³ C NMR (75 MHz, DMSO-d₆) δ14.0, 38.7, 41.6, 61.5, 112.9, 117.7,119.8, 130.3, 142.2, 160.2, 168.9.

EXAMPLE III Preparation of a Reagent of General Formula I EthylN-Iodoacetyl-4-aminomethylsalicylate ##STR19## EthylN-Chloroacetyl-4-aminomethylsalicylate

Ethyl 4-aminomethylsalicylate hydrochloride (0.50 g, 2.17 mmoles) wassuspended in dry N,N-diethylformamide (25 mL) andN,N-diisopropylethylamine (0.38 mL, 2.18 mmoles) was added. Once theamine salt dissolved, chloroacetic anhydride (0.39 g, 2.25 mmoles) wasadded and the reaction mixture was stirred at room temperature for 4.5hours. The reaction mixture was then diluted with ethyl acetate (100mL), and this solution was extracted with 1N aqueous hydrochloric acid(100 mL), water (50 mL), and saturated aqueous sodium chloride (50 mL).The ethyl acetate solution was dried over anhydrous magnesium sulfate,filtered and evaporated to dryness to afford a white solid. Finally,this solid was crystallized from ethyl acetate:hexanes (8:2, 10 mL) toafforded 0.13 g (22% yield) of ethylN-chloroacetyl-4-aminomethylsalicylate (m.p. 120°-121° C.).

¹ H NMR (300 MHz, DMSO-d₆) δ1.31, (triplet, J=7 Hz, 3H, CH₂ CH₃), 4.14(singlet, 2H, ClCH₂), 4.29 (doublet, J=6 Hz, 2H, NHCH₂), 4.34 (quartet,J=7 Hz, 2H, CH₂ CH₃), 6.82 (doublet, J=8 Hz, 1H, ArH), 6.84 (singlet,1H, ArH), 7.73 (doublet, J=8 Hz, 1H, ArH), 8.78 (triplet, J=6 Hz, 1H,NH), 10.58 (singlet, 1H, OH). ¹³ C NMR (75 MHz, DMSO-d₆) δ13.9, 42.1,42.6, 61.3, 111.6, 115.6, 118.4, 130.1, 147.6, 160.6, 166.5, 169.1.

Ethyl N-Iodoacetyl-4-aminomethylsalicylate

Ethyl N-chloroacetyl-4-aminomethylsalicylate (0.11 g, 0.40 mmoles) wasdissolved in acetone (5 mL) and sodium iodide (0.06 g, 0.40 mmoles) wasadded. The solution was refluxed for 2.5 hours, then cooled to roomtemperature and filtered. The filtrate was evaporated to dryness toafford a white solid of ethyl N-iodoacetyl-4-aminomethylsalicylate (0.19g, 100% yield) (m.p. 105°-108° C.).

¹ H NMR (300 MHz, DMSO-d₆) δ1.31, (triplet, J=7 Hz, 3H, CH₂ CH₃), 3.68(singlet, 2H, ICH₂), 4.26 (doublet, J=6 Hz, 2H, NHCH₂), 4.33 (quartet,J=7 Hz, 2H, CH₂ CH₃), 6.82 (doublet, J=8 Hz, 1H, ArH), 6.84 (singlet,1H, ArH), 7.72 (doublet, J=8 Hz, 1H, ArH), 8.79 (triplet, J=6 Hz, 1H,NH), 10.58 (singlet, 1H, OH). ¹³ C NMR (75 MHz, DMSO-d₆) δ0.41, 13.9,42.0, 61.3, 111.4, 115.5, 118.3, 130.1, 147.9, 160.6, 168.3, 169.1.

EXAMPLE III Preparation of a Reagent of General Formula I Ethyl(6-Aminohexanoyl)aminomethylsalicylate Trifluoroacetate ##STR20## Ethyl(N-tert-Butoxycarbonyl-6-aminohexanoyl)aminomethylsalicylate

Ethyl 4-aminomethylsalicylate hydrochloride (0.52 g, 1.28 mmoles) wassuspended in anhydrous N,N-dimethylformamide (25 mL), andN,N-diisopropylethylamine (0.79 mL, 4.53 mmoles) was added, followed byN-tert-butoxycarbonyl-6-aminohexanoic acid succinimidyl ester (0.74 g,2.26 mmoles). The mixture was stirred under dry nitrogen for 18 hours,during which time all solids dissolved. The reaction mixture was dilutedwith ethyl acetate (100 mL) and extracted with 1N aqueous hydrochloricacid (100 mL). The layers were separated, and the ethyl acetate solutionwas washed with water (100 mL) and saturated aqueous sodium chloride(500 mL). The ethyl acetate solution was dried over anhydrous magnesiumsulfate, filtered, and evaporated to afford an amorphous off-whitesolid. Finally, the solid was crystallized from ethyl acetate, filtered,and dried in vacuo to afford 0.67 g (73% yield) of ethyl(N-tert-butoxycarbonyl-6-aminohexanoyl)aminomethylsalicylate (m.p.120°-121° C., open capillary, uncorrected).

¹ H NMR (300 MHz, DMSO-d₆) δ1.19 (multiplet, 2H, NHCH₂ CH₂ CH₂ CH₂ CH₂CO), 1.34 (multiplet, 5H, CH₂ CH₂ CO and CH₂ CH₃), 1.34 (singlet, 9H,C(CH₃)₃), 1.49 (multiplet, 2H, NHCH₂ CH₂), 2.12 (triplet, J=7 Hz, 2H,CH₂ CH₂ CO), 2.87 (quartet, J=6 Hz, 2H, NHCH₂ CH₂), 4.23 (doublet, J=6Hz, 2H, CH₂ Ar), 4.32 (quartet, J=7 Hz, CH₂ CH₃), 6.74 (triplet, J=6 Hz,1H, CONHCH₂ CH₂), 6.80 (doublet, J=8 Hz, 1H, ArH), 6.81 singlet, 1H,ArH), 7.71 (doublet, J=8 Hz, 1H, ArH), 8.34 (triplet, J=6 Hz, 1H,CONHCH₂ Ar), 10.58 (singlet, 1H, OH). ¹³ C NMR (75 MHz, DMSO-d₆) δ14.0,25.1, 26.3, 28.3, 29.7, 36.3, 40.2, 42.9, 61.4, 79.0, 111.6, 116.0,118.3, 130.4, 146.9, 156.2, 161.9, 170.1, 173.0.

Ethyl (6-Aminohexanoyl)aminomethylsalicylate Trifluoroacetate

Ethyl (N-tert-butoxycarbonyl-6-aminohexanoyl)aminomethylsalicylate (0.58g, 1.41 mmoles) was dissolved in dichloromethane (5 mL) and the solutionwas cooled in an ice/water bath. Trifluoroacetic acid (5 mL) was added,and the reaction was allowed to warm to room temperature. After 2 hours,the reaction mixture was evaporated to dryness to give the product as anoil, which was dried in vacuo over potassium hydroxide pellets to afford0.59 g (99% yield) of ethyl (6-aminohexanoyl)aminomethylsalicylatetrifluoroacetate.

¹ H NMR (300 MHz, DMSO-d₆) δ1.28 (multiplet, 5H, H₃ CH₂ CH₂ CH₂ CH₂ CH₂CO and CH₂ CH₃), 1.53 (multiplet, 4H, NH₃ CH₂ CH₂ CH₂ CH₂ CH₂ CO), 2.15(triplet, J=8 Hz, 2H, CH₂ CH₂ CO), 2.71 (multiplet, 2H, NH₃ CH₂ CH₂),4.21 (doublet, J=6 Hz, 2H, CH₂ Ar), 4.30 (quartet, J=8 Hz, 2H, CH₂ CH₃),6.79 (doublet, J=8 Hz, 1H, ArH), 6.81 (singlet, 1H, ArH), 7.68 (doublet,J=8 Hz, 1H, ArH), 8.18 (broad singlet, 3H, NH₃), 8.60 (triplet, J=6 Hz,1H, CONHCH₂ Ar), 10.58 (broad singlet 1H, OH). ¹³ C NMR (75 MHz,DMSO-d₆) δ14.0, 24.8, 25.6, 26.7, 35.1, 38.6, 41.7, 61.3, 111.5, 115.5,118.3, 130.1, 148.6, 160.6, 169.2, 172.6.

EXAMPLE IV Preparation of a Reagent of General Formula I Methyl4Succinylaminomethylsalicylate Succinimidyl Ester ##STR21## Methyl4-Methylsalicylate

4-Methylsalicylic acid (100 g, 658 mmoles) was dissolved in anhydrousmethanol (500 mL) and concentrated sulfuric acid (25 mL) was addedcarefully. The solution was refluxed for 18 hours, then cooled to roomtemperature. The reaction mixture was concentrated to about 150 mL, andethyl acetate (250 mL) was added. The ethyl acetate solution was washedtwice with saturated aqueous sodium bicarbonate (250 mL portions) andthen with saturated aqueous sodium chloride (100 mL). The ethyl acetatesolution was dried over anhydrous sodium sulfate, filtered, andevaporated to a clear, reddish-brown liquid. This crude product wasvacuum distilled (oil pump) to afford a clear, viscous liquid thatsolidified on standing to afford 98.1 g (90% yield) of methyl4-methylsalicylate.

¹ H NMR (300 MHz, CHCl₃ -d) δ2.32 (singlet, 3H, ArCH₃), 3.91 (singlet,3H, OCH₃), 6.67 (doublet, J=8 Hz, 1H, ArH), 6.78 (singlet, 1H, ArH),7.69 (doublet, J=8 Hz, 1H, ArH), 10.71 (singlet, 1H, OH). ¹³ C NMR (75MHz, CHCl3-d) δ21.8, 52.1, 110.0, 117.9, 120.6, 129.9, 147.3, 161.9,170.9.

Methyl 4-Bromomethylsalicylate

Methyl 4-methylsalicylate (98.1 g, 590 mmoles) was dissolved in carbontetrachloride (600 mL), and N-bromosuccinimide (105.0 g, 590 mmoles) andbenzoyl peroxide (0.7 g, 3 mmoles) were added. The mixture was refluxedunder nitrogen. After 2 hours, an additional portion (0.7 g) ofN-bromosuccinimide was added. Reflux was continued for 16 hours. Thereaction mixture was cooled to room temperature and the solid removed byfiltration. The yellow filtrate was evaporated to dryness to afford athick yellow syrup that solidified on standing. Hexanes (500 mL) wasadded to the solid, and the mixture was boiled until almost all solidsdissolved. The hot hexanes solution was filtered and concentrated untila solid just began to precipitate. The mixture was heated to dissolvethe solid, and the solution was allowed to cool slowly to roomtemperature. Pale yellow crystals formed slowly. The mixture was thenchilled in ice for 2 hours to complete crystallization. Finally, thesolid was filtered, washed with cold hexanes (100 mL), and dried invacuo to afford 83.5 g (58% yield) of methyl 4-bromomethylsalicylate(m.p. 73°-75° C., open capillary, uncorrected).

¹ H NMR (300 MHz, CHCl₃ -d) δ3.95 (singlet, 3H, OCH₃), 4.40 (singlet,2H, CH₂), 6.90 (doublet, J=8 Hz, 1H, ArH), 7.00 (singlet, 1H, ArH), 7.80(doublet, J=8 Hz, 1H, ArH), 10.78 (singlet, 1H, OH). ¹³ C NMR (75 MHz,CHCl₃ -d) δ32.1, 52.5, 112.4, 118.2, 120.1, 130.7, 145.8, 162.0, 170.5.

Methyl 4-Azidomethylsalicylate

Methyl 4-bromomethyl salicylate (83.5 g, 341 mmoles) was dissolved indry N,N-dimethylformamide (150 mL) and sodium azide 925.0 g, 380 mmoles)was added. The yellow suspension was stirred at room temperature, andthe solids rapidly dissolved. The solution turned brown, and aprecipitate of sodium bromide formed. The reaction mixture was stirred16 hours, then filtered. The filtrate was evaporated to a brown oil,which was dissolved in a mixture of hexanes and ethyl acetate (1:1 v/v!.100 mL). Silica gel (25 g, flash chromatography grade) was added to thebrown solution, and the mixture was swirled well. The silica was removedby filtration on a glass frit, and washed three times with hexanes:ethylacetate (1:1 v/v!, 50 mL portions). The silica gel was dried on thefrit, and the combined filtrates were evaporated to dryness to afford adark yellow liquid. The crude product (which was utilized for thefollowing reaction) was found to contain some residualN,N-dimethylformamide.

¹ H NMR (300 MHz, CHCl₃ -d) δ3.94 (singlet, 3H, OCH₃), 4.32 (singlet,2H, CH₂), 6.82 (doublet, J=8 Hz, 1H, ArH), 6.93 (singlet, 1H, ArH), 7.83(doublet, J=8 Hz, 1H, ArH), 10.80 (singlet, 1H, OH). ¹³ C NMR (75 MHz,CHCl₃ -d) 52.5, 54.3, 112.3, 117.0, 118.7, 130.8, 143.9, 162.1, 170.5.

Methyl 4-Aminomethylsalicylate Hydrochloride

Crude methyl 4-azidomethylsalicylate was dissolved in methanol (750 mL)in a 2 L Parr hydrogenation flask. Palladium on carbon catalyst (10%w/w!, 3.8 g) in water (25 mL) was added, followed by concentratedhydrochloric acid (35 mL). The flask was affixed to a Parr hydrogenator,and the mixture was shaken at room temperature under 40 psi of hydrogenfor 16 hours. The reaction mixture was then filtered through a 0.45 mmnylon filter. The retained solid was then washed with methanol (150 mL),water (100 mL), and methanol again (150 mL). The combined filtrates wereevaporated to dryness to afford a tan solid. This solid was dissolved inhot denatured ethanol (150 mL) and the solution was allowed to cool toroom temperature. White crystals formed quickly. Finally, the mixturewas then chilled for 16-18 hours at 4° C. to complete crystallization.The solid was filtered, washed with a little cold ethanol (50 mL) andthen diethyl ether (150 mL), and dried in vacuo over potassium hydroxidepellets to afford 51.5 g (65% yield based on methyl4-bromomethylsalicylate) of methyl 4-aminomethylsalicylate hydrochloride(m.p. 225°-227° C., open capillary, uncorrected).

¹ H (300 MHz, DMSO-d₆) δ3.87 (singlet, 3H, OCH₃), 4.00 (singlet, 2H,CH₂), 7.06 (doublet, J=8 Hz, 1H, ArH), 7.13 (singlet, 1H, ArH), 7.77(doublet, J=8 Hz, 1H, ArH), 8.59 (broad singlet, 3H, NH₃ Cl), 10.55(singlet, 1H, OH). ¹³ C NMR (75 MHz, DMSO-d₆) δ41.6, 52.6, 113.0, 117.7,119.8, 130.4, 142.1, 160.0, 169.1

Methyl 4-Succinylaminomethylsalicylate

Methyl 4-aminomethylsalicylate hydrochloride (3.00 g, 13.8 mmoles) wassuspended in dry pyridine (25 mL), and N,N-diisopropylethylamine (2.7mL, 15.5 mmoles) was added. The suspension was stirred in an ice/waterbath for 15 minutes, and then succinic anhydride (1.36 g, 13.6 mmoles)was added. The mixture was allowed to warm to room temperature, duringwhich time the starting solids dissolved. After stirring for 2 hours,the mixture was evaporated to dryness, and the resulting solid waspartitioned between ethyl acetate (300 mL) and 1M aqueous hydrochloricacid (100 mL). The layers were separated, and the ethyl acetate solutionwas washed with 1M hydrochloric acid (100 mL) and saturated aqueoussodium chloride (100 mL). The solution was dried over anhydrous sodiumsulfate, filtered, and evaporated to dryness to yield a white solid.Finally, the solid was crystallized from ethyl acetate/hexanes,filtered, and dried in vacuo to afford 3.02 g (87% yield) of methyl4-succinylaminomethylsalicylate (m.p. 161°-163° C.).

¹ H NMR (300 MHz, DMSO-d₆) δ2.43 (triplet, J=6 Hz, 2H, CH₂ CONH), 2.49(triplet, J=6 Hz, 2H, HO₂ CCH₂), 3.87 (singlet, 3H, OCH₃), 4.27(doublet, J=6 Hz, 2H, ArCH₂ NH), 6.83 (doublet, J=8 Hz, 1H, ArH), 6.86(singlet, 1H, ArH), 7.71 (doublet, J=8 Hz, 1H, ArH), 8.43 (triplet, J=6Hz, 1H, NH), 10.54 (singlet, 1H, OH), 12.12 (singlet, 1H, CO₂ H). ¹³ CNMR (75 MHz, DMSO-d₆) δ29.1, 30.1, 41.9, 52.5, 111.3, 115.6, 118.4,130.1, 148.7, 160.7, 169.7, 171.7, 174.2.

Methyl 4-Succinylaminomethylsalicylate Succinimidyl Ester

Methyl 4-succinylaminomethylsalicylate (2.60 g, 10.2 mmoles) wasdissolved in dry tetrahydrofuran (100 mL), and N-hydroxysuccinimide(1.29 g, 11.2 mmoles) and 1,3-di-cyclohexylcarbodiimide (2.10 g, 10.2mmoles) were added. The mixture was stirred at room temperature underdry nitrogen, and the solids rapidly dissolved. After about 20 minutes,a white precipitate formed. The reaction mixture was stirred 16-18hours, then chilled several hours at -20° C. The mixture was filteredcold, and the solid washed with a little tetrahydrofuran (25 mL). Thecombined filtrates were evaporated to dryness, and the residue wascrystallized from ethyl acetate/hexanes, filtered, and dried in vacuo toafford 2.39 g (62% yield) of methyl 4-succinyl-aminomethylsalicylatesuccinimidyl ester (m.p. 133°-135° C.).

¹ H NMR (300 MHz, DMSO-d₆) δ2.56 (triplet, J=7 Hz, 2H, CH₂ CONH), 2.80(singlet, 4H, COCH₂ CH₂ CO), 2.91 (triplet, J=7 Hz, 2H, NO₂ CCH₂), 3.87(singlet, 3H, OCH₃), 4.27 (doublet, J=6 Hz, 2H, ArCH₂ NH), 6.83(doublet, J=9 Hz, 1H, ArH), 6.84 (singlet, 1H, ArH), 7.71 (doublet, J=9Hz, 1H, ArH), 8.51 (triplet, J=6 Hz, 1H, NH), 10.50 (singlet, 1H, OH).¹³ C NMR (75 MHz, DMSO-d₆) δ525.4, 25.9, 29.2, 41.8, 52.4, 111.4, 115.6,118.4, 130.2, 148.2, 160.4, 168.9, 169.4, 170.2, 170.4.

EXAMPLE V Preparation of a Reagent of General Formula I Methyl(6-Aminohexanoyl)aminomethylsalicylate Hydrochloride ##STR22## Methyl(N-tert-Butoxycarbonyl-6-aminohexanoyl)aminomethylsalicylate

Methyl 4-aminomethylsalicylate hydrochloride (2.25 g, 10.3 mmoles) wassuspended in anhydrous N,N-dimethylformamide (30 mL), andN,N-diisopropylethylamine (3.6 mL, 20.7 mmoles) was added, followed byN-tert-butoxycarbonyl-6-aminohexanoic acid succinimidyl ester (3.38 g,10.3 mmoles). The mixture was stirred under dry nitrogen for 18 hours,during which time all solids dissolved. The solvent was then evaporatedto leave a light brown syrup, which was partitioned between ethylacetate (100 mL) and 1M aqueous hydrochloric acid (100 mL). The layerswere separated, and the ethyl acetate solution was washed with saturatedaqueous sodium bicarbonate (100 mL) and saturated aqueous sodiumchloride (100 mL). The ethyl acetate solution was dried over anhydroussodium sulfate, filtered, and evaporated to an amorphous off-whitesolid. The solid was crystallized form ethyl acetate/hexanes, filtered,and dried in vacuo to afford 3.25 g (80% yield) of methyl(N-tert-butoxycarbonyl-6-aminohexanoyl)aminomethylsalicylate (m.p.120°-121° C., open capillary, uncorrected).

¹ H NMR (300 MHz, DMSO-d₆) δ1.22 (multiplet, 2H, NHCH₂ CH₂ CH₂ CH₂ CH₂CO), 1.36 (multiplet, 2H, CH₂ CH₂ CO), 1.36 (singlet, 9H, C(CH₃)₃), 1.51(multiplet, 2H, NHCH₂ CH₂), 2.13 (triplet, J=7 Hz, 2H, CH₂ CH₂ CO), 2.87(quartet, J=6 Hz, 2H, NHCH₂ CH₂), 3.87 (singlet, 3H, OCH₃), 4.24(doublet, J=6 Hz, 2H, CH₂ Ar), 6.75 (triplet, J=6 Hz, 1H, CONHCH₂ CH₂),6.80 (doublet, J=8 Hz, 1H, ArH), 6.82 (singlet, 1H, ArH), 7.72 (doublet,J=8 Hz, 1H, ArH), 8.35 (triplet, J=6 Hz, 1H, CONHCH₂ Ar), 10.53(singlet, 1H, OH). ¹³ C NMR (75 MHz, CHCl₃ -d) δ25.0, 26.0, 28.2, 29.3,35.3, 41.7, 52.4, 77.4,111.2, 115.5, 118.3, 130.1, 148.7, 155.8, 160.5,169.5, 172.6.

Methyl (6-Aminohexanoyl)aminomethylsalicylate Hydrochloride

Methyl (N-tert-butoxycarbonyl-6-aminohexanoyl)aminomethylsalicylate(3.00 g, 7.60 mmoles) was dissolved in ethyl acetate (100 mL), and dryhydrogen chloride was bubbled slowly through the solution. The reactionmixture warmed as the gas dissolved. After 5 minutes, the gas was shutoff, and the solution was stirred at room temperature. A whiteprecipitate formed in the solution. After 30 minutes, the reactionmixture was chilled in ice for 2 hours, then the solid was filtered,washed with diethyl ether (50 mL) and dried in vacuo over potassiumhydroxide pellets to afford 2.50 g (99% yield) of methyl(6-aminohexanoyl)aminomethylsalicylate hydrochloride (decomposes witheffervescence at 158°-160° C., open capillary, uncorrected).

¹ H NMR (300 MHz, DMSO-d₆) δ1.28 (multiplet, 2H, H₃ CH₂ CH₂ CH₂ CH₂ CH₂CO), 1.54 (multiplet, 4H, NH₃ CH₂ CH₂ CH₂ CH₂ CH₂ CO), 2.16 (triplet,J=8 Hz, 2H, CH₂ CH₂ CO), 2.71 (multiplet, 2H, NH₃ CH₂ CH₂), 3.85(singlet, 3H, OCH₃), 4.22 (doublet, J=6 Hz, 2H, CH₂ Ar), 6.80 (doublet,J=8 Hz, 1H, ArH), 6.83 (singlet, 1H, ArH), 7.70 (doublet, J=8 Hz, 1H,ArH), 8.10 (broad singlet, 3H, NH₃), 8.54 (triplet, J=6 Hz, 1H, CONHCH₂Ar), 10.35 (broad singlet 1H, OH). ¹³ C NMR (75 MHz, CHCl₃ -d) δ24.8,25.6, 26.7, 35.1, 41.7, 111.5, 115.6, 118.4, 130.2, 148.6, 160.4, 169.4.

EXAMPLE VI Preparation of a Reagent of General Formula I Cyanomethyl4-Glutarylaminomethylsalicylate Succinimidyl Ester ##STR23## MethylN-tert-Butoxycarbonylaminomethylsalicylate

Methyl 4-aminomethylsalicylate hydrochloride (10.9 g, 50 mmoles) wassuspended in anhydrous methanol (200 mL) and di-tert-butyldicarbonate(10.9 g, 50 mmoles) and triethylamine (7.0 mL, 50 mmoles) were added.The solid rapidly dissolved with the slow evolution of gas. The reactionmixture was stirred at room temperature for 18 hours under dry nitrogen,then evaporated to dryness to afford a white amorphous mass. This masswas partitioned between ethyl acetate (200 mL) and water (100 mL). Thelayers were separated, and the ethyl acetate solution was dried overanhydrous sodium sulfate. The solution was filtered and evaporated to awhite solid. This solid was crystallized from ethyl acetate/hexanes,filtered, and dried in vacuo to afford 13.7 g (97% yield) of methylN-tert-butoxycarbonylaminomethyl-salicylate (m.p. 95°-96° C., opencapillary, uncorrected).

¹ H NMR (300 MHz, CHCl₃ -d) δ1.42 (singlet, 9H, C(CH₃)₃), 3.90 (singlet,3H, OCH₃), 4.26 (doublet, J=6 Hz, 2H, CH₂ Ar), 4.99 (triplet, J=6H, 1H,NH), 6.75 (doublet, J=8 Hz, 1H, ArH), 6.84 (singlet, 1H, ArH), 7.73(doublet, J=8 Hz, 1H, ArH), 10.72 (singlet, 1H, OH). ¹³ C NMR (75 MHz,CHCl₃ -d) δ28.5, 44.4, 52.4, 80.0, 111.5, 115.9, 118.2, 130.5, 150.0,156.3, 162.1, 170.8.

N-tert-Butoxycarbonylaminomethylsalicylic acid

Methyl N-tert-butoxycarbonylaminomethylsalicylate (8.7 g, 30.9 mmoles)was dissolved in dry tetrahydrofuran (100 mL), and potassiumtrimethylsilanolate (4.4 g, 30.9 mmoles, 90% pure) was added. The yellowsolution was refluxed for 24 hours, during which time a tan precipitateformed and the solvent turned light brown. The mixture was evaporated todryness, and the solid was dissolved in cold water (100 mL). The brownsolution was chilled in an ice bath, and saturated aqueous potassiumhydrogen sulfate solution was used to titrate the stirred solution to pH2-3. An off-white solid precipitated during the titration. The solid wasfiltered, washed with cold water, and dried in vacuo over potassiumhydroxide to afford 6.7 g, (81% yield) of crudeN-tert-butoxycarbonylaminomethylsalicylic acid (m.p. 141°-144° C.,decomposes with effervescence on melting, open capillary, uncorrected).

¹ H NMR (300 MHz, CHCl₃ -d) δ1.47 (singlet, 9H, C(CH₃)₃), 4.33 (doublet,J=6 Hz, 2H, CH₂ Ar), 5.07 (triplet, J=6H, 1H, NH), 6.80 (doublet, J=8Hz, 1H, ArH), 6.88 (singlet, 1H, ArH), 7.81 (doublet, J=8 Hz, 1H, ArH),10.72 (broad singlet, 2H, OH and CO₂ H). ¹³ C NMR (75 MHz, CHCl₃ -d)δ28.5, 44.4, 80.5, 111.1, 115.9, 118.3, 131.5, 148.5, 156.6, 162.6,174.1.

Cyanomethyl N-tert-Butoxycarbonylaminomethylsalicylate

N-tert-Butoxycarbonylamino-methylsalicylic acid (8.2 g, 30.6 mmoles) wassuspended in chloroacetonitrile (25 mL), and triethylamine (4.3 mL, 30.6mmoles) was added. The mixture was stirred under dry nitrogen at 50° C.,and the solids dissolved. The solution was stirred 18 hours, and thencooled to room temperature. The solvent was evaporated, and the residuewas partitioned between ethyl acetate (250 mL) and water (250 mL). Thelayers were separated, and the ethyl acetate layer was washed withsaturated aqueous sodium bicarbonate (100 mL) and saturated aqueoussodium chloride (100 mL). The solution was dried over anhydrous sodiumsulfate, filtered, and evaporated to dryness. The residual pale tansolid was dissolved in ethyl acetate (100 mL), and silica gel (10 g,flash chromatography grade) was added. The mixture was swirled well andallowed to sit for five minutes at room temperature. The silica wasremoved by filtration on a glass frit, and washed with ethyl acetate(100 mL). The filtrate was evaporated to dryness. The solid residue wascrystallized from ethyl acetate/hexanes to afford 7.9 g (88% yield) ofcyanomethyl N-tert-butoxycarbonylaminomethylsalicylate (m.p. 144°-146°C., open capillary, uncorrected).

¹ H NMR (300 MHz, CHCl₃ -d) δ1.45 (singlet, 9H, C(CH₃)₃), 4.30 (doublet,J=6 Hz, 2H, CH₂ Ar), 5.00 (singlet, 2H, OCH₂ CN), 5.05 (triplet, J=6H,1H, NH), 6.83 (doublet, J=8 Hz, 1H, ArH), 6.91 (singlet, 1H, ArH), 7.77(doublet, J=8 Hz, 1H, ArH), 10.12 (singlet, 1H, OH). ¹³ C NMR (75 MHz,CHCl₃ -d) δ28.4, 44.3, 49.0, 80.1, 109.6, 114.2, 116.1, 118.7, 130.5,149.7, 156.2, 162.5, 168.5.

Cyanomethyl Aminomethylsalicylate Hydrochloride

Cyanomethyl N-tert-butoxycarbonylaminomethylsalicylate (7.7 g, 26.2mmoles) was dissolved in tetrahydrofuran (150 mL), and dry hydrogenchloride was bubbled slowly through the solution. The reaction mixturewarmed as the gas dissolved. After 5 minutes, the gas was shut off, andthe solution was stirred at room temperature. A thick, creamy whiteprecipitate formed in the solution. After 30 minutes, the reactionmixture was chilled in ice for 2 hours, then the solid was filtered,washed with diethyl ether (100 mL) and dried in vacuo over potassiumhydroxide pellets to afford 5.8 g (91% yield) of cyanomethylaminomethylsalicylate hydrochloride (m.p. darkens at 210° C., decomposesat 228° C., open capillary, uncorrected).

¹ H NMR (300 MHz, DMSO-d6) δ4.00 (singlet, 2H, CH₂ Ar), 5.20 (singlet,2H, OCH₂ CN), 7.05 (doublet, J=8 Hz, 1H, ArH), 7.15 (singlet, 1H, ArH),7.75 (doublet, J=8 Hz, 1H, ArH), 8.62 (broad singlet, 3H, NH₃), 10.38(singlet, 1H, OH). ¹³ C NMR (75 MHz, DMSO-d6) δ41.6,49.7, 113.0, 116.1,118.0, 119.7, 131.1, 142.3, 159.5, 166.1.

Cyanomethyl 4-Glutarylaminomethylsalicylate

Cyanomethyl 4-aminomethylsalicylate hydrochloride (1.22 g, 5.0 mmoles)was suspended in dry dichloromethane (100 mL), and the suspension wasstirred in an ice/water bath. A solution of glutaric anhydride (0.57 g,5.0 mmoles) and triethylamine (0.7 mL, 5.0 mmoles) in drydichloromethane (25 mL) was then added dropwise over 15 minutes. Themixture was allowed to warm to room temperature, and the reaction wasstirred for 18 hours. The mixture was evaporated to dryness, and theresulting solid was triturated under cold 0.1M aqueous hydrochloric acid(50 mL). The solid was collected by filtration, washed with cold water,and dried in vacuo over potassium hydroxide pellets to afford 1.43 g(89% yield) of cyanomethyl 4-glutarylaminomethylsalicylate (m.p.125°-126° C.).

¹ H NMR (300 MHz, DMSO-d₆) δ1.75 (quintet, J=7 Hz, 2H, CH₂ CH₂ CH₂),2.19 (triplet, J=7 Hz, 2H, CH₂ CONH), 2.22 (triplet, J=7 Hz, 2H, HO₂CCH₂), 4.24 (doublet, J=6 Hz, 2H, ArCH₂ NH), 5.17 (singlet, 2H, OCH₂CN), 6.81 (doublet, J=8 Hz, 1H, ArH), 6.85 (singlet, 1H, ArH), 7.70(doublet, J=8 Hz, 1H, ArH), 8.39 (triplet, J=6 Hz, 1H, NH), 10.5 (verybroad singlet, 1H, OH), 11.7 (very broad singlet, 1H, CO₂ H). ¹³ C NMR(75 MHz, DMSO-d₆) δ20.7, 33.1, 34.5, 41.8, 49.7, 111.2, 115.9, 116.2,118.5, 130.9, 149.1, 160.2, 166.6, 172.3, 174.5.

Cyanomethyl 4-Glutarylaminomethy salicylate Succinimidyl Ester

Cyanomethyl 4-glutarylaminomethylsalicylate (1.00 g, 3.1 mmoles) wasdissolved in dry tetrahydrofuran (50 mL), and N-hydroxysuccinimide (0.36g, 3.1 mmoles) and 1,3-cyclohexylcarbodiimide (0.64 g, 3.1 mmoles) wereadded. The mixture was stirred at room temperature under dry nitrogen,and the solids rapidly dissolved. After about 60 minutes, a whiteprecipitate formed. The reaction mixture was stirred 24 hours, thenchilled several hours at -20° C. The mixture was filtered cold, and thesolid washed with a little tetrahydrofuran (10 mL). The combinedfiltrates were evaporated to dryness, and the residue was crystallizedfrom ethyl acetate/hexanes, filtered, and dried in vacuo to afford 1.04g (80% yield) of cyanomethyl 4-glutarylaminomethylsalicylatesuccinimidyl ester (m.p. 116°-119° C.).

¹ H NMR (300 MHz, DMSO-d₆) δ1.88 (quintet, J=7 Hz, 2H, CH₂ CH₂ CH₂),2.30 (triplet, J=7 Hz, 2H, CH₂ CONH), 2.71 (triplet, J=7 Hz, 2H, NO₂CCH₂), 2.80 (singlet, 4H, COCH₂ CH₂ CO), 4.26 (doublet, J=6 Hz, 2H,ArCH₂ NH), 5.18 (singlet, 2H, OCH₂ CN), 6.82 (doublet, J=8 Hz, 1H, ArH),6.86 (singlet, 1H, ArH), 7.71 (doublet, J=8 Hz, 1H, ArH), 8.44 (triplet,J=6 Hz, 1H, NH), 10.18 (broad singlet, 1H, OH). ¹³ C NMR (75 MHz,DMSO-d₆) δ20.4, 25.5, 29.7, 33.6, 41.8, 49.6, 111.2, 115.9, 116.2,118.4, 130.9, 148.9, 160.1, 166.5, 169.0, 170.5, 171.7.

EXAMPLE VII Preparation of a Reagent of General Formula I Cyanomethyl4-(6-maleimidohexanoyl)aminiomethylsalicylate ##STR24## Cyanomethyl4-(6-maleimidohexanoyl)aminomethylsalicylate

Cyanomethyl 4-aminomethylsalicylate hydrochloride (79 mg, 0.32 mmoles)and 6-maleimidocaproic acid N-hydroxysuccinimide ester (100 mg, 0.32mmoles) were suspended in dry N,N-dimethylformamide (5.0 mL), and thesuspension was stirred at room temperature. N,N-diisopropylethylamine(87 μL, 0.50 mmoles) was added. The solids rapidly dissolved to afford aclear, pale yellow solution. After 30 minutes, the mixture wasevaporated to dryness, and the residue was partitioned between ethylacetate (25 mL) and cold 0.1M aqueous hydrochloric acid (25 mL). Thelayers were separated, and the ethyl acetate solution washed withsaturated aqueous sodium bicarbonate (25 mL) and saturated aqueoussodium chloride (25 mL). The ethyl acetate solution was dried overanhydrous sodium sulfate, filtered and evaporated to a white solid. Thesolid was crystallized from ethyl acetate/hexanes to afford 108 mg (84%yield) of cyanomethyl 4-(6-maleimidohexanoyl)aminomethyl salicylate(m.p. 141°-144° C.).

¹ H NMR (300 MHz, DMSO-d₆) δ1.20 (multiplet, 2H, CH₂ CH₂ CH₂ CH₂ CH₂),1.53 (multiplet, 4H, CH₂ CH₂ CH₂ CH₂ CH₂), 2.13 (triplet, J=7 Hz, 2H,NCH₂ CH₂), 3.38 (triplet, J=7 Hz, 2H, CH₂ CH₂ CO), 4.26 (doublet, J=6Hz, 2H, ArCH₂ NH), 5.02 (singlet, 2H, OCH₂ CN), 6.68 (singlet, 2H,CH═CH), 6.76 (doublet, J=8 Hz, 1H, ArH), 6.80 (singlet, 1H, ArH), 7.69(doublet, J=8 Hz, 1H, ArH), 7.99 (triplet, J=6 Hz, 1H, NH), 10.04(singlet, 1H, OH). ¹³ C NMR (75 MHz, DMSO-d₆) δ24.5, 25.7, 27.6, 35.2,36.9, 41.9, 48.8, 109.1, 114.3, 115.6, 118.2, 129.8, 133.8, 149.2,161.2, 167.6, 170.4, 172.7.

EXAMPLE VII Preparation of a Reagent of General Formula I Cyanomethyl4-(3-(2-Pyridyldithio)propionyl)aminomethylsalicylate ##STR25##Cyanomethyl 4-(3-(2-pyridyldithio)propionyl)aminomethylsalicylate

Cyanomethyl 4-aminomethylsalicylate hydrochloride (79 mg, 0.32 mmoles)and 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (100mg, 0.32 mmoles) were suspended in dry N,N-dimethylformamide (5.0 mL),and the suspension was stirred at room temperature.N,N-Diisopropylethylamine (87 μL, 0.50 mmoles) was added. The solidsrapidly dissolved to give a clear, pale tan solution. After 30 minutes,the mixture was evaporated to dryness, and the residue was partitionedbetween ethyl acetate (25 mL) and cold 0.1M aqueous hydrochloric acid(25 mL). The layers were separated and the ethyl acetate solution washedwith saturated aqueous sodium bicarbonate (25 mL) and saturated aqueoussodium chloride (25 mL).

The ethyl acetate solution was dried over anhydrous sodium sulfate,filtered and evaporated to a clear oil. Trituration under cold hexanesafforded 64 mg (48% yield) of a gum of cyanomethyl4-(3-(2-pyridyldithio)propionyl)aminomethylsalicylate.

¹ H NMR (300 MHz, DMSO-d₆) δ2.60 (triplet, J=7 Hz, 2H, SCH₂ CH₂), 3.05(triplet, J=7 Hz, 2H, CH₂ CH₂ CO), 4.27 (doublet, J=6 Hz, 2H, ArCH₂ NH),5.19 (singlet, 2H, OCH₂ CN), 6.84 (doublet, J=8 Hz, 1H, ArH), 6.88(singlet, 1H, ArH), 7.23 (triplet, J=6 Hz, 1H, ArH), 7.71 (doublet, J=8Hz, 1H, ArH), 7.74-7.82 (multiplet, 2H, ArH), 8.44 (doublet, J=6 Hz, 1H,ArH), 8.56 (triplet, J=6 Hz, 1H, NH), 10.13 (broad singlet, 1H, OH). ¹³C NMR (75 MHz, DMSO-d₆) δ34.0, 34.5, 41.8, 49.6, 54.9, 111.2, 115.9,116.0, 118.4, 119.3, 121.3, 130.8, 137.9, 148.6, 149.7, 159.4, 160.0,166.4, 170.3.

EXAMPLE IX Preparation of a Reagent of General Formula III4-Glutarylaminomethylsalicylhydroxamic Acid Hydrazide ##STR26## Methyl4-Glutarylaminomethylsalicylate N-tert-Butyloxycarbonylhydrazide

Methyl 4-glutarylaminomethylsalicylate succinimidyl ester (2.57 g, 6.8mmoles) prepared as for the succinyl derivative above, substitutingglutaric anhydride for succinic anhydride! was dissolved in drytetrahydrofuran (100 mL), and tert-butylcarbazate (0.90 g, 6.8 mmoles)was added. The reaction was stirred at room temperature for 60 hours.The solution was then evaporated to dryness, and the residue dissolvedin ethyl acetate (100 mL). The ethyl acetate solution was washed withsaturated aqueous potassium bicarbonate (100 mL) and saturated aqueoussodium chloride (100 mL). It was then dried over anhydrous sodiumsulfate, filtered, and evaporated to dryness. The residue wascrystallized by dissolving it in ethyl acetate (50 mL) with gentlewarming, adding hexanes (50 mL) to the warm solution, and chilling at-20° C. Once crystallization began, another portion of hexanes (50 mL)was added, and the mixture was chilled for 18 hours at -20° C. Finally,the solid was filtered, washed with hexanes:ethyl acetate (2:1 v/v!, 90mL), and dried in vacuo to afford 2.51 g (90% yield) of methyl4-glutarylaminomethylsalicylate N-tert-butyloxycarbonylhydrazide (m.p.68°-72° C., open capillary, uncorrected).

¹ H NMR (300 MHz, DMSO-d₆) δ1.37 (singlet, 9H, (CH₃)₃ C), 1.74 (quintet,J=7 Hz, 2H, CH₂ CH₂ CH₂), 2.07 (triplet, J=7 Hz, 2H, CH₂ CONHCH₂), 2.17(triplet, J=7 Hz, 2H, HNHNOCCH₂), 3.86 (singlet, 3H, OCH₃), 4.23(doublet, J=6 Hz, 2H, ArCH₂ NH), 6.80 (doublet, J=8 Hz, 1H, ArH), 6.82(singlet, 1H, ArH), 7.71 (doublet, J=8 Hz, 1H, ArH), 8.37 (triplet, J=6Hz, 1H, NH), 8.65 (singlet, 1H, NHNHCOCH₂), 9.48 (singlet, 1H, OCONHNH),10.50 (singlet, 1H, OH). ¹³ C NMR (75 MHz, DMSO-d₆) δ21.2, 28.0, 32.6,34.6, 41.7, 52.4, 79.1, 111.4, 115.5, 118.3, 130.2, 148.5, 155.5, 160.4,169.4, 171.7, 172.1.

4-Glutarylaminomethylsalicylhydroxamic AcidN-tert-Butyloxycarbonylhydrazide

Methyl 4-glutarylaminomethylsalicylate N-tert-butyloxycarbonylhydrazide(2.00 g, 4.9 mmoles) was added to a cooled (ice/water bath) solution ofhydroxylamine sulfate (0.82 g, 5.0 mmoles), sodium hydroxide (1.00 g,25.0 mmoles) and sodium sulfite (0.20 g, mmoles) in water (25 mL). Thesuspension was stirred for 18 hours in the dark, allowing it to warm toroom temperature. The solution was then filtered to remove someinsoluble material, and the pale yellow solution was chilled in anice/water bath. The cold reaction mixture was slowly titrated to pH 3-4with cold sulfuric acid (25% v/v! aqueous), during which time a gummymaterial precipitated. The mixture was then chilled several hours inice, and the liquid was decanted. The remaining amorphous solid wasdissolved in methanol (25 mL), filtered, and evaporated to a white foam.The foam was dried in vacuo to 1.86 g (94% yield) of crude4-glutarylaminomethylsalicylhydroxamic acidN-tert-butyloxycarbonylhydrazide.

¹ H NMR (300 MHz, DMSO-d₆) δ1.37 (singlet, 9H, (CH₃)₃ C), 1.74 (quintet,J=7 Hz, 2H, CH₂ CH₂ CH₂), 2.10 (triplet, J=7 Hz, 2H, CH₂ CONHCH₂), 2.16(triplet, J=7 Hz, 2H, HNHNOCCH₂), 4.20 (doublet, J=6 Hz, 2H, ArCH₂ NH),6.71 (doublet, J=8 Hz, 1H, ArH), 6.74 (singlet, 1H, ArH), 7.60 (doublet,J=8 Hz, 1H, ArH), 8.32 (triplet, J=6 Hz, 1H, NH), 8.65 (singlet, 1H,NHNHCOCH₂), 9.29 (broad singlet, 1H, NHOH), 9.48 (singlet, 1H, OCONHNH),11.37 (broad singlet, 1H, ArOH), 12.32 (broad singlet, 1H, NHOH). ¹³ CNMR (75 MHz, DMSO-d₆) δ21.2, 28.0, 32.6, 34.6, 41.7, 79.1, 112.4, 115.6,117.6, 127.1, 145.9, 155.6, 159.8, 166.5, 171.7, 172.1.

4-Glutarylaminomethylsalicylhydroxamic Acid Hydrazide Hydrochloride

4-Glutarylaminomethylsalicylhydroxamic acidN-tert-butyloxycarbonylhydrazide (1.86 g, 4.6 mmoles) was dissolved indry tetrahydrofuran (100 mL), and dry hydrogen chloride was bubbledslowly through the solution. The reaction mixture warmed as the gasdissolved. After 5 minutes, the gas was shut off, and the solution wasstirred at room temperature. A white precipitate formed in the solution.After 30 minutes, the reaction mixture was chilled in ice for 2 hours,then the solid was filtered, washed with diethyl ether (50 mL) and driedin vacuo over potassium hydroxide pellets to afford 1.51 g (95% yield)of 4-glutarylaminomethylsalicylhydroxamic acid hydrazide hydrochloride(m.p. shrinks at 65° C., decomposes with effervescence at 100° C., opencapillary, uncorrected).

¹ H NMR (300 MHz, DMSO-d₆) δ1.72 (quintet, J=7 Hz, 2H, CH₂ CH₂ CH₂),2.17 (triplet, J=7 Hz, 2H, CH₂ CONHCH₂), 2.23 (triplet, J=7 Hz, 2H, H₃NHNOCCH₂), 4.17 (doublet, J=6 Hz, 2H, ArCH₂ NH), 6.69 (doublet, J=8 Hz,1H, ArH), 6.74 (singlet, 1H, ArH), 7.66 (doublet, J=8 Hz, 1H, ArH), 8.49(triplet, J=6 Hz, 1H, NH), 10.49 (broad singlet, 4H, NH₃ NHCOCH₂), 11.08(broad singlet, 1H, ArOH), 11.43 (broad singlet, 2H, NHOH). ¹³ C NMR (75MHz, DMSO-d₆) δ21.0, 25.2, 32.3, 34.4, 41.8, 67.2, 112.6, 115.7, 117.7,127.4, 145.9, 159.8, 166.5, 171.7, 172.1.

EXAMPLE IX Preparation Of A Reagent Of General Formula III4-Glutarylaminomethylsalicyl(O-methyl)hydroxamic Acid Succinimidyl Ester##STR27## Methyl N-(Benzyloxycarbonyl)-4-aminomethylsalicylate

Methyl 4-aminomethylsalicylate hydrochloride (5.04 g, 23.2 mmoles) wassuspended in chloroform (80 mL) and N,N-diisopropylethylamine (4.10 mL,23.5 mmoles) and N-(benzyloxycarbonyloxy)succinimide (6.48 g, 26.0mmoles) were added. The reaction mixture was stirred at room temperaturefor 4 hours, during which time all solids dissolved. The reactionmixture was then extracted with 1N aqueous hydrochloric acid (100 mL),water (75 mL), and saturated aqueous sodium chloride (50 mL). Thechloroform solution was dried over anhydrous magnesium sulfate, filteredand evaporated to dryness to give a white solid. The product wascrystallized from ethyl acetate:hexanes to afford 6.17 g (84% yield) ofmethyl N-(benzyloxycarbonyl)4-aminomethylsalicylate (m.p. 91°-92° C.,open capillary, uncorrected).

¹ H NMR (300 MHz, DMSO-d₆) δ3.86 (singlet, 3H, OCH₃), 4.21 (doublet, J=6Hz, 2H, NHCH₂), 5.05 (singlet, 2H, CH₂ O), 6.82 (doublet, J=8 Hz, 1H,ArH), 6.86 (singlet, 1H, ArH), 7.27-7.36 (multiplet, 5H, ArH), 7.72(doublet, J=8 Hz, 1H, ArH), 7.90 (triplet, J=6 Hz, 1H, NH), 10.53(singlet, 1H, OH). ¹³ C NMR (75 MHz, DMSO-d₆) δ43.5, 52.4, 65.6, 111.5,115.4, 118.2, 127.9, 128.0, 128.5, 130.2, 137.3, 148.6, 156.7, 160.5,169.5.

Methyl N-(Benzyloxycarbonyl)-4-aminomethyl-2-O-benzylsalicylate

Methyl N-(benzyloxycarbonyl)-4-aminomethylsalicylate (6.06 g, 19.2mmoles) was dissolved in acetone (150 mL), and benzyl bromide (2.60 mL,21.9 mmoles) and anhydrous potassium carbonate (13.28 g, 96.1 mmoles)were added. The mixture was stirred and heated at reflux for 22 hours.The mixture was concentrated to remove most of the acetone, and ethylacetate (100 mL) was added. Aqueous hydrochloric acid (1N, 200 mL) wasadded slowly, swirling frequently to dissolve the solid carbonate. Thelayers were separated, and the aqueous layer was extracted with ethylacetate (50 mL). The ethyl acetate solutions were combined and washedwith water (100 mL) and saturated aqueous sodium chloride (50 mL), driedover anhydrous magnesium sulfate, filtered, and concentrated to about 50mL. This solution was heated to boiling, and hexanes (150 mL) wereadded. The solution was cooled in ice, and crystals slowly formed. Thecrystals were collected by filtration, washed with hexanes, and dried invacuo to afford 6.97 g (89% yield) of methylN-(benzyloxycarbonyl)-4-aminomethyl-2-O-benzylsalicylate (m.p. 106°-107°C., open capillary, uncorrected).

¹ H NMR (300 MHz, DMSO-d₆) δ3.78 (singlet, 3H, OCH₃), 4.27 (doublet, J=6Hz, 2H, NHCH₂), 5.07 (singlet, 2H, CH₂ OCO), 5.15 (singlet, 2H, CH₂ O),6.94 (doublet, J=8 Hz, 1H, ArH), 7.16 (singlet, 1H, ArH), 7.26-7.42(multiplet, 8H, ArH), 7.49-7.51 (multiplet, 2H, ArH), 7.68 (doublet, J=8Hz, 1H, ArH), 7.90 (triplet,J=6 Hz, 1H, NH) ¹³ C NMR (75 MHz, DMSO-d₆)δ43.8, 51.8, 65.6, 69.7, 112.5, 112.6, 118.9, 119.0, 127.2, 127.9,128.0, 128.5 (2 carbons), 131.3, 137.0, 137.3, 146.2, 156.7, 157.8,166.1.

N-(Benzyloxycarbonyl)-4-aminomethyl-2-O-benzylsalicylic acid

Methyl N-(benzyloxycarbonyl)4-aminomethyl-2-O-benzylsalicylate (6.81 g,16.8 mmoles) was dissolved in tetrahydrofuran (50 mL). A solution oflithium hydroxide monohydrate (0.78 g, 18.5 mmoles) in water (25 mL) wasadded, and the reaction mixture was stirred and heated at 75° C. for 24hours. The solution was cooled, and 1N aqueous hydrochloric acid (50 mL)was added. The reaction mixture was extracted twice with ethyl acetate(150 mL then 50 mL). The combined ethyl acetate extracts were washedwith water (75 mL) and saturated aqueous sodium chloride (50 mL), driedover anhydrous magnesium sulfate, filtered, and concentrated to 150 mL.The ethyl acetate solution was heated to boiling, and hexanes (150 mL)was added. The solution was cooled in ice, and crystals formed. Thecrystals were collected by filtration, washed with hexanes, and dried invacuo to afford 5.92 g (90% yield) ofN-(benzyloxy-carbonyl)-4-aminomethyl-2-O-benzylsalicylic acid (m.p.139°14 140° C., open capillary, uncorrected).

¹ H NMR (300 MHz, DMSO-d₆) δ4.23 (doublet, J=6 Hz, 2H, NHCH₂), 5.06(singlet, 2H, CH₂ OCO), 5.13 (singlet, 2H, CH₂ O), 6.90 (doublet, J=8Hz, 1H, ArH), 7.11 (singlet, 1H, ArH), 7.28-7.40 (multiplet, 8H, ArH),7.48-7.51 (multiplet, 2H, ArH), 7.64 (doublet, J=8 Hz, 1H, ArH), 7.88(triplet, J=6 Hz, 1H, NH), 12.57 (singlet, 1H, COOH). ¹³ C NMR (75 MHz,DMSO-d₆) δ43.8, 65.6, 69.8, 112.5 (2 carbons), 118.9, 120.4, 127.4,127.9, 128.0, 128.5 (2 carbons), 131.2, 137.1, 137.4, 145.6, 156.7,157.5, 167.4.

N-(Benzyloxycarbonyl)-4-aminomethyl-2-O-benzylsalicyl(O-methyl)hydroxamicacid

N-(Benzyloxycarbonyl)-4-aminomethyl-2-O-benzylsalicylic acid (3.07 g,7.84 mmoles) was dissolved in anhydrous N,N-dimethylformamide (75 mL)under dry nitrogen. After cooling the solution to in an ice/water bath,triethylamine (2.20 mL, 15.8 mmoles) was added followed by isobutylchloroformate (1.10 mL, 8.48 mmoles). The reaction was stirred in icefor 1.5 hours. Methoxylamine hydrochloride (0.67 g, 8.02 mmoles) wasadded and the reaction mixture was allowed to warm to room temperature.After 3 hours, the reaction was diluted with ethyl acetate (150 mL) andextracted with 1N aqueous hydrochloric acid (100 mL), water (100 mL),and saturated aqueous sodium chloride (50 mL). The ethyl acetatesolution was dried over anhydrous magnesium sulfate, filtered andconcentrated to about 50 mL. This solution was boiled and hexanes (100mL) were added. Upon cooling in an ice bath, crystals formed quickly.They were collected by filtration and washed with hexane to afford 2.87g (87% yield) ofN-benzyloxycarbonyl)-4-aminomethyl-2-O-benzylsalicyl(O-methyl)hydroxamicacid (m.p. 116°-117° C., open capillary, uncorrected).

¹ H NMR (300 MHz, DMSO-d₆) δ3.63 (singlet, 3H, NHOCH₃), 4.20 (doublet,J=6 Hz, 2H, NHCH₂), 5.04 (singlet, 2H, CH₂ OCO), 5.13 (singlet, 2H, CH₂O), 6.90 (doublet, J=8 Hz 1H, ArH), 7.08 (singlet, 1H, ArH), 7.29-7.49(multiplet, 11H, ArH), 7.88 (triplet, J=6 Hz, 1H, NH), 11.13 (singlet,1H, NHOCH₃). ¹³ C NMR (75 MHz, DMSO-d₆) δ43.7, 63.2, 65.5, 69.8, 111.8,119.2, 121.7, 127.6, 127.9, 128.0, 128.1, 128.5 (2 carbons), 129.8,136.8, 137.3, 144.3, 155.9, 156.6, 163.3.

4-Aminomethylsalicyl(O-methyl)hydroxamic Acid Hydrochloride

N-(Benzyloxycarbonyl)-4-aminomethyl-2-O-benzylsalicyl(O-methyl)hydroxamicacid (1.42 g, 3.38 mmoles) and palladium on carbon (0.10 g, 10% w/w!)were placed in a 1 L hydrogenation flask under dry nitrogen. Ethanol(150 mL) was added, followed by concentrated aqueous hydrochloric acid(0.30 mL). The flask was affixed to the Parr hydrogenator and shakenunder 35 psi of hydrogen for 7 hours at room temperature. The reactionmixture was then filtered through Celite to remove the catalyst, and thefiltrate was concentrated until a precipitate began to form. The mixturewas cooled in ice, the solid collected by filtration, washed withhexanes, and dried in vacuo to afford 0.65 g (83% yield) of4-aminomethyl-salicyl(O-methyl)hydroxamic acid hydrochloride (m.p. >250°C., open capillary, uncorrected).

¹ H NMR (300 MHz, DMSO-d₆) δ3.71 (singlet, 3H, NHOCH₃), 3.95 (singlet,2H, NH₃ CH₂), 7.00 (doublet, J=8 Hz, 1H, ArH), 7.05 (singlet, 1H, ArH),7.72 (doublet, J=8 Hz, H, ArH), 8.53 (broad singlet, 3H, NH₃), 12.01(broad singlet, 2H, NHOCH₃ and OH). ¹³ C NMR (75 MHz, DMSO-d₆) δ41.7,63.6, 114.2, 117.6, 119.3, 128.2, 140.0, 159.4, 166.1.

4-Glutarylaminomethylsalicyl(O-methyl)hydroxamic Acid Succinimidyl Ester

4-Aminomethylsalicyl(O-methyl)hydroxamic acid hydrochloride (0.57 g,1,82 mmoles) was dissolved in anhydrous N,N-dimethylformamide (10 mL),and N,N-diisopropylethyl amine (0.35 mL, 2.01 mmoles) and glutaricanhydride (0.23 g, 2.02 mmoles) were added. The mixture was stirred atroom temperature for 26 hours, and then N-hydroxysuccinimide (0.23 g,2.00 mmoles) and 1,3-dicyclohexylcarbodiimide (0.42 g, 2.01 mmoles) wereadded. The reaction mixture was stirred for an additional 24 hours atroom temperature. The mixture was then filtered and diluted with ethylacetate (100 mL). The ethyl acetate solution was washed with 1N aqueoushydrochloric acid (100 mL), water (100 mL), and saturated aqueous sodiumchloride (50 mL). The solution was then dried over anhydrous magnesiumsulfate, filtered, and evaporated to dryness. The crude gummy productwas triturated under a mixture of ethyl acetate and diethyl ether toproduce a solid which was filtered and dried in vacuo to afford 0.15 g(20% yield) of 4-glutarylaminomethylsalicyl(O-methyl)hydroxamic acidsuccinimidyl ester (m.p. 121°-124° C., open capillary, uncorrected).

¹ H NMR (300 MHz, DMSO-d₆) δ1.85 (quintet, J=7 Hz, 2H, CH₂ CH₂ CH₂),2.27 (triplet, J=7 Hz, 2H, CH₂ CONHCH₂), 2.69 (triplet, J=7 Hz, 2H,NOCCH₂), 2.80 (singlet, 4H, COCH₂ CH₂ CO), 3.70 (singlet, 3H, NHOCH₃),4.21 (doublet, J=6 Hz, 2H, NHCH₂), 6.75 (doublet, J=8 Hz, 1H, ArH), 6.77(singlet, 1H, ArH), 7.57 (doublet, J=8 Hz, H, ArH), 8.39 (triplet, J=6Hz, 1H, NHCH₂), 11.75 (singlet, 1H, OH), 11.78 (singlet, 1H, NHOCH₃). ¹³C NMR (75 MHz, DMSO-d₆) δ20.4, 25.4, 29.7, 33.4, 33.6, 41.7, 112.8,115.6, 117.8, 127.8, 146.2, 159.3, 166.3, 169.0, 170.5, 171.5.

EXAMPLE XI Preparation Of A Reagent Of General Formula III4-(3-(2-Pyridyldithio)propionyl)aminomethylsalicyl(O-methyl)hydroxamicAcid ##STR28##4-(3-(2-Pyridyldithio)propionyl)aminomethylsalicyl(O-methyl)hydroxamicAcid

3-(2-Pyridyldithio)propionic acid N-hydroxysuccinimide ester (100 mg,0.32 mmole) was dissolved in dry N,N-dimethylformamide (5.0 mL) andN,N-diisopropylethylamine (65 μL, 0.36 mmole) was added followed by4-aminomethylsalicyl(O-methyl)hydroxamic acid hydrochloride (82 mg, 0.35mmole). The reaction was stirred for 6 hours at room temperature. Thesolvent was evaporated in vacuo, and the residue was chromatographed onsilica gel eluting with dichloromethane/methanol/acetic acid (95:5:1v/v/v!). Fractions containing the desired product were pooled andevaporated to an oil to afford 44 mg (35% yield) of4-(3-(2-pyridyldithio)propionyl)aminomethylsalicyl(O-methyl)hydroxamicacid.

¹ H NMR (300 MHz,DMSO-d₆) δ2.58 (triplet, J=7 Hz, 2H, CH₂ CH₂ S), 3.04(triplet, J=7 Hz, 2H, COCH₂ CH₂), 3.70 (singlet, 3H, OCH₃), 4.23(doublet, J=6 Hz, 2H, ArCH₂ NH), 6.76 (doublet, J=8 Hz, 1H, ArH), 6.79(singlet, 1H, ArH), 7.23 (triplet, J=6 Hz, 1H, ArH), 7.57 (doublet, J=8Hz, 1H, ArH), 7.73-7.82 (multiplet, 2H, ArH), 8.44 (doublet, J=6 Hz, 1H,ArH), 8.48 (triplet, J=6 Hz, 1H, NH), 11.80 (broad singlet, 2H, OH andNHO). ¹³ C NMR (75 MHz, DMSO-d₆) δ34.1, 34.5, 41.8, 63.5, 112.7, 115.7,117.8, 119.3, 121.3, 127.7, 138.0, 145.9, 149.8, 159.4, 166.3, 170.2,172.8.

PHENYLBORONIC ACID COMPLEXING REAGENTS AND CONJUGATES DERIVED FROM4-AMINOMETHYL-2,6-DIHYDROXYBENZOIC ACID

A three-step process which utilizes reagents of General Formula CI forthe preparation of bioconjugates is summarized in FIG. 1'. Initially, areagent of General Formula CI is selected that is comprised of anappropriate reactive electrophilic or nucleophic group R₁ suitable forreaction with the desired biologically active species. ##STR29##

Group R₁ is a reactive electrophilic or nucleophilic moiety suitable forreaction of the putative phenylboronic acid complexing reagent with abioactive species. Group R₁ is preferably selected from, but not limitedto, acrylamide, bromo, dithiopyridyl, bromoacetamide, hydrazide,N-hydroxysuccinimidyl ester, N-hydroxysulfosuccinimidyl ester, imidateester, imadazolide, iodo, iodoacetamide, maleimide, amino and thiolmoieties.

Group Z is a spacer selected from a saturated or unsaturated, preferablyunbranched, chain of from about 0 to 6 carbon equivalents in length, anunbranched saturated or unsaturated chain of from about 6 to 18 carbonequivalents in length with at least one of intermediate amide ordisulfide moieties, and a polyethylene glycol chain of from about 3 to12 carbon equivalents in length. Group Z is preferably selected from anunbranched alkyl chain of general formula (CH₂)_(n), wherein n=1 to 6.

Group R₂ is selected from an alkyl (e.g., methyl, ethyl, etc.) and amethylene bearing an electronegative substituent. An electronegativesubstituent is a substituent with a negative dipole moment, e.g., CN,COOH, etc. Group R₂ is preferably selected from one of CH₃, CH₂ CH₃, CH₂CN, CH₂ COOH, CH₂ CONH₂ and CH₂ OCH₃.

The next step in a three-step process in the preparation ofbioconjugates is to condense the appropriate reagent with the bioactivespecies to yield a conjugate of General Formula CII: ##STR30## InGeneral Formula CII, Z and R₂ are as defined above, and BAS represents abiologically active species which may or may not contain a portion of areactive moiety (and any spacer) used to attach the biologically activespecies to the reagent.

Next, the conjugate is reacted with a hydroxylamine derivative of thegeneral formula NH₂ OR₃, wherein R₃ is selected from either an H, analkyl (e.g., methyl, ethyl, etc.), or a methylene or ethylene with anelectronegative moiety. Suitable hydroxylamine derivatives include, butnot limited to, NH₂ OH, NH₂ OCH₃, NH₂ OCH₂ CN, NH₂ OCH₂ COOH, NH₂ OCH₂CONH₂ and NH₂ OCH₂ CH₂ OH. The resulting conjugate has the generalformula of General Formula CIV: ##STR31## In General Formula CIV, GroupsZ and BAS are as defined above, and R₃ is selected from one of an H, analkyl, and a methylene moiety with an electronegative substituent.

The conjugate of General Formula CIV is then complexed with aphenylboronic acid having the general formula of General Formula CV:##STR32## wherein the symbol labeled BAS* represents a secondbiologically active species, that may include a linker portion anddiffer from the biologically active species labeled BAS of thecomplexing reagent. The BAS* may also include a portion of a reactivemoiety used to attach the bioactive species to the phenylboronic acidreagent. The complexation yields the stereoisomeric complex (abouttetrahedral boron) of General Formula CVI: ##STR33##

It will be appreciated by one of ordinary skill in the art, uponreference to this disclosure, that the various syntheses described abovein the previous section (concerning reagents and conjugates derived from4 or 5 aminomethyl-salicylic acid and shown in FIGS. 1 through 7) may beutilized where appropriate for the corresponding synthesis of thereagents shown in FIGS. 1'-7' and/or described in this section. Forexample, the techniques for adding Z groups and R1 groups from theprevious section may be considered for use in preparing the reagentsshown in FIGS. 1' through 7' and/or described in this section.

SYNTHESIS OF PUTATIVE PHENYLBORONIC ACID COMPLEXING REAGENTS OF GENERALFORMULA CI ##STR34##

Reagents of General Formula CI are derived from alkyl2,6-dihydroxy-4-methylbenzoates. FIG. 2' summarizes the preparation ofalkyl 4-aminomethyl-2,6-dihydroxybenzoates, synthetic intermediatesleading to reagents of General Formula CI, wherein R₂ is an alkyl group,e.g., methyl, ethyl, etc. FIG. 2' shows an example where R₂ is a methylgroup. Initially, in step 1, an alkyl 2,6dihydroxy-4-methylbenzoate isacetylated to reduce the reactivity of the ring system towardbromination, affording the corresponding alkyl2,6-diacetoxy-4-methylbenzoate. In step 2, the ester is brominated withN-bromosuccinimide and benzoyl peroxide catalyst to afford thecorresponding benzyl bromide. In step 3, the benzyl bromide is alkylatedwith sodium azide to afford the corresponding benzyl azide. In step 4,the benzyl azide is subjected to palladium catalyzed hydrogenation inthe presence of HCl to afford the corresponding benzyl aminehydrochloride. Finally, in step 5, the acetoxy protecting groups areremoved by acid catalyzed methanolysis.

FIG. 3' summarizes the preparation of synthetic intermediates leading toreagents of General Formula CI, wherein group R₂ is a methylene bearingan electronegative moiety, for example, carboxymethyl, acetamidomethyland cyanomethyl 4-aminomethyl-2,6-dihydroxybenzoate. It is to beappreciated that other contemplated substituents for group R₂ may beprepared by a similar synthesis. Initially, in step 1, the alkyl4-aminomethyl-2,6-dihydroxy-benzoate, prepared as summarized in FIG. 2',is reacted with di-tert-butyl dicarbonate and triethylamine in methanolto afford the corresponding protected alkylN-(tert-butoxycarbonyl)aminomethylbenzoate. In step 2, the alkyl esteris cleaved by reaction with potassium trimethylsilanolate and worked upin aqueous acid to afford the corresponding benzoic acid. In step 3, thebenzoic acid is alkylated by reaction with either an α-haloacid,α-haloacetamide or α-haloacetonitrile and triethylamine to afford thecorresponding carboxymethyl, acetamidomethyl or cyanomethyl ester,respectively. Finally, in step 4, the N-(tert-butoxycarbonyl) protectinggroup is removed by reaction with anhydrous HCl in tetrahydrofuran toafford an alkyl 4-aminomethyl-2,6-dihydroxybenzoate hydrochloride.

Another reagent of the present invention, which is the acrylic acidamide of alkyl 4-amino methyl 2,6 dihydroxy-benzoate can be prepared ina single step, using the end product of FIG. 3 by condensing acrylicacid anhydride or acryloyl chloride with alkyl 4-aminomethyl 2,6dihydroxy-benzoate.

FIG. 5 ' summarizes the synthesis of reagents of General Formula CI,wherein group R₂ is one of either an alkyl or a methylene bearing anelectronegative moiety, and wherein group R₁ is selected from eitherimidazolide, hydrazide and N-hydroxysuccinimidyl ester moieties. Thesereagents are each prepared by a two-step process in which an aliphaticacid anhydride is utilized in the first step. Initially, an alkyl4-aminomethyl-2,6-dihydroxybenzoate, prepared as summarized in eitherFIG. 2' or FIG. 3', is condensed of an aliphatic acid anhydridepreferably selected from, but not limited to, either succinic anhydride,glutaric anhydride, maleic anhydride and glycolic acid anhydride, in anaprotic organic solvent, which results in the introduction of a spacer(group Z) having a fire terminal carboxylic acid moiety. In the casewhere the aliphatic acid anhydride is maleic anhydride in thiscondensation reaction, the resulting Z group is unsaturated as itcontains an alkene group. Subsequently, the carboxylic acid moiety isfurther functionalized by reaction with either N,N-carbonyldiimidazole,isobutylchloroformate and tert-butyl carbazate, orN,N-dicyclohexylcarbodiimide and N-hydroxy-succinimide to afford thecorresponding imidazolide, protected hydrazide andN-hydroxy-succinimidyl ester, respectively. In the instance of theprotected hydrazide, the N-(tert-butoxycarbonyl) protecting group isremoved by contacting the reagent with anhydrous hydrochloric acid.

FIG. 6' summarizes the synthesis of reagents of General Formula CI,wherein group R₂ is one of either an alkyl or a methylene bearing anelectronegative moiety, and wherein group R₁ is selected from eitheracrylamide, bromoacetamide, chloroacetamide, maleimide, dithiopyridyland imidate ester moieties. Reagents of General Formula CI, whereingroup R₁ is selected from either bromo and chloro moieties, are preparedby condensing an alkyl 4- or 5-aminomethyl-salicylate, prepared assummarized in either FIG. 2' or FIG. 3', with either bromoacetic acidanhydride or chloroacetic acid anhydride, respectively. The homologousiodo reagent is prepared by halogen exchange of the chloro reagent withsodium iodide. Reagents of General Formula CI, wherein R₁ is selectedfrom either bromo, chloro, iodo, bromoacetamide, chloroacetamide andiodoacetamide moieties, may not be conveniently prepared when R₃ is H,due to the potential for intermolecular alkylation of the unprotectedhydroxamate. Reagents of General Formula CI, wherein group R₁ isselected from either maleimide and dithiopyridyl moieties, are preparedby condensing an alkyl 4-aminomethyl-2,6-dihydroxybenzoate, prepared assummarized in either FIG. 2' or FIG. 3', with an N-hydroxysuccinimidylester of an aliphatic carboxylic ester which bears either a terminalmaleimide or dithiopyridyl moiety. Reagents of General Formula CI,wherein R₁ is an imidate ester moiety, are prepared by a two-stepprocess in which an alkyl 4-aminomethyl-2,6-dihydroxybenzoate, preparedas summarized in either FIG. 2' or FIG. 3', is first condensed with anN-hydroxysuccinimidyl ester of an aliphatic carboxylic ester which bearsa terminal nitrile moiety. Subsequently, the nitrile moiety is convertedto the methyl imidate ester by reaction with anhydrous hydrogen chlorideacid in methanol at 0° C.

Reagents of General Formula CI, wherein group R₁ is selected from eitherN-hydroxysuccinimidyl ester and dithiopyridyl moieties, may be utilizedas synthetic intermediates to prepare reagents of General Formula CI,wherein group Z is an unbranched saturated or unsaturated chain with atleast one of intermediate amide or disulfide moieties.

Reagents of General Formula CI, wherein group R₁ is anN-hydroxysuccinimidyl ester moiety, may be condensed with compoundshaving primary aliphatic amine moieties of the general formula R₁ --Z₂--NH₂, wherein Z₂ is an unbranched saturated or unsaturated chainpreferably of from about 1 to 5 carbon equivalents in length, to affordreagents of General Formula CI, wherein group Z is an unbranchedsaturated or unsaturated chain with at least one of intermediate amidemoieties.

Alternatively, N-hydroxysuccinimidyl ester reagents of General FormulaCI, preferably derived from a dicarboxylic acid selected from eithersuccinic acid, maleic acid, fumaric acid, acetylenedicarboxylic acid andglutaric acid, may be condensed with compounds having primary aliphaticamine moieties of the general formula HO₂ C--Z₂ --NH₂, wherein Z₂ is anunbranched saturated or unsaturated chain preferably of from about 1 to5 carbon equivalents in length, preferably selected from, but notlimited to, either glycine, β-alanine, aminopropiolic acid,4-aminobutyric acid and 6-aminocaproic acid, to afford compounds havingfree terminal carboxylic acid moieties which may be furtherfunctionalized in accordance with FIG. 5' to afford reagents of GeneralFormula CI, wherein Z is an unbranched saturated or unsaturated chainwith at least one of intermediate amide moieties. This process issummarized in FIG. 7' for the synthesis of a reagent of General FormulaCI, wherein R₁ is an N-hydroxysuccinimidyl ester, and wherein Z is anunbranched saturated or unsaturated chain with at least one of anintermediate amide moiety.

Reagents of General Formula CI, wherein group R₁ is a dithiopyridylmoiety, may be condensed with compounds having terminal thiol moietiesof the general formula R₁ --Z₂ --SH, wherein Z₂ is an unbranchedsaturated or unsaturated chain preferably of from about 1 to 5 carbonequivalents in length, to afford reagents of General Formula CI, whereingroup Z is an unbranched saturated or unsaturated chain with at leastone of intermediate disulfide moieties.

Alternatively, dithiopyrinyl reagents of General Formula CI, preferablyderived from a mercaptocarboxylic acid selected from eithermercaptoacetic acid, β-mercaptopropionic acid, mercaptopropiolic acid,4-mercaptobutyric acid and 6-mercaptocaproic acid, may be condensed withcompounds having a thiol moiety of the general formula HO₂ C--Z₂ --SH,wherein Z₂ is an unbranched saturated or unsaturated chain preferably offrom about 1 to 5 carbon equivalents in length, preferably selectedfrom, either mercaptoacetic acid, β-mercaptopropionic acid,mercaptopropiolic acid, 4-mercaptobutyric acid and 6-mercaptocaproicacid, to afford compounds having free terminal carboxylic acid moietieswhich may be further functionalized in accordance with FIG. 5' to affordreagents of General Formula I, wherein group Z is an unbranchedsaturated or unsaturated chain with at least one of intermediatedisulfide moieties. This process is summarized in FIG. 7' for thesynthesis of a reagent of General Formula CI, wherein R₁ is anN-hydroxysuccinimidyl ester, and wherein Z is an unbranched saturated orunsaturated chain with at least one of an intermediate disulfide moiety.

Reagents of General Formula CI, wherein group Z is a polyethylene glycolchain of from about 3 to 12 carbon equivalents in length, are preparedby condensing an alkyl 4-aminomethyl-2,6-dihydroxybenzoate, prepared assummarized in either FIG. 2' or FIG. 3', with a polyethylene glycolreagent having both an N-hydroxysuccinimidyl ester moiety and either areactive electrophilic or nucleophilic moiety (or a protected precursorthereof), many of which are commercially available, to afford reagentsof General Formula CI, wherein group Z is a polyethylene glycol chain offrom about 3 to 12 carbon equivalents in length.

SYNTHESIS OF PHENYLBORONIC ACID COMPLEXING REAGENTS OF GENERAL FORMULACIII ##STR35##

FIG. 4' summarizes the preparation of4-aminomethyl-2,6-dihydroxybenzohydroxamic acids, syntheticintermediates leading to reagents of General Formula CIII, wherein groupR₃ is one of an alkyl or methylene bearing an electronegative moiety.Initially, in step 1, an alkyl 4-aminomethyl-2,6-dihydroxybenzoate,prepared as summarized in FIG. 2', is condensed withN-(benzyloxycarbonyl)oxy succinimide to afford the alkylN-benzyloxycarbonyl protected alkyl 4-aminomethyl-2,6-dihydroxybenzoate.In step 2, the phenolic hydroxyl moieties are condensed with benzylbromide to afford the further protected benzyl ether intermediate. Instep 3, the all ester is selectively cleaved by reaction with LiOH toafford the corresponding carboxylic acid. In step 4, the carboxylic acidis activated by reaction with isobutylchloroformate to form a mixedanhydride which is subsequently reacted with a hydroxylamine derivativepreferably selected from, but not limited to, either NH₂ OH, NH₂ OCH₃,NH₂ CH₂ CN, NH₂ OCH₂ COOH, NH₂ OCH₂ CONH₂, NH₂ OCH₂ CH₂ OH and NH₂ OCH₂OCH₃ to afford the corresponding protected hydroxamic acid. Finally, instep 5, both the amine and phenolic hydroxyl moieties are simultaneouslydeprotected by paladium catalyzed hydrogenation in the presence of HClto afford the corresponding 4-aminomethyl-2,6-dihydroxybenzohydroxamicacid hydrochloride. These synthetic intermediates, which arestructurally related to the corresponding alkyl4amino-methyl-2,6-dihydroxybenzoates, may be further functionalized inaccordance with either FIG. 5' or FIG. 6' to afford reagents of GeneralFormula CIII.

PREPARATION OF PHENYLBORONIC ACID COMPLEXING CONJUGATES OF GENERALFORMULA CIV

At this point, the putative phenylboronic acid complexing reagents ofGeneral Formula CI may be reacted with a suitable biologically activespecies to yield the conjugate of the general formula of General FormulaCII: ##STR36##

The conjugate of General Formula CII is next condensed with a hydroxylamine derivative to yield the corresponding phenylboronic acidcomplexing conjugate of the general formula of General Formula CIV:##STR37##

Suitable hydroxylamine derivatives are preferably selected from, but notlimited to, NH₂ OH, NH₂ OCH₃, NH₂ OCH₂ CN, NH₂ OCH₂ COOH, NH₂ OCH₂CONH₂, NH₂ OCH₂ CH₂ OH, NH₂ OCH₂ OCH₃. When group R₂ in General FormulaCII is an alkyl group, NH₂ OH is preferably utilized to effect theinterconversion of General Formula CII to General Formula CIV.

Alternatively, conjugates of General Formula CIV may also be prepared bythe route which is also described in FIG. 1' which utilizes a reagent ofGeneral Formula CIII: ##STR38##

Phenylboronic acid complexing reagents of General Formula CIII may bereacted with a suitable biologically active species to yield theconjugate of the general formula of General Formula CIV: ##STR39##

PREPARATION OF BIOCONJUGATES OF GENERAL FORMULA CVI

Bioconjugates of General Formula CVI may be prepared in buffered aqueoussolutions or organic solvents. Preferred buffers include acetate,citrate, phosphate, carbonate and diglycine. Borate buffers should beavoided due to their ability to complex with the phenylboronic acidcomplexing moiety. Tris, β-hydroxyamine and β-hydroxyacid buffers shouldbe avoided due to their ability to complex with the phenylboronic acid.The bioconjugate is formed within a few minutes over a range oftemperatures of from about 4° C. to 70° C. The stability of thebioconjugate in aqueous solution at a given pH and temperature isinfluenced, to some extent, by substituent group R₃. Bioconjugates ofGeneral Formula CVI are stable in aqueous solutions of approximate pHgreater than 2.5 and less than 12.5. The bioconjugation reaction(phenylboronic acid complexation) is insensitive to significantvariations in ionic strength over the range 0.01 to 1M, the presence oforganic solvents including acetonitrile, methanol, ethanol, isopropanol,butanol, N,N-dimethylformamide and dimethylsulfoxide, the presence ofdetergents, and the presence of chaotropic agents (protein denaturants)including urea, guanidine hydrochloride, guanidine thiocyanate andformamide, which are incompatible with prior art indirect labelingsystems wherein the structure of a biological macromolecule must bemaintained to preserve requisite binding properties. Once formed, thebioconjugates are stable upon removal of water, and can be lyophilizedfor storage.

The stability of the bioconjugate at a given pH is determined to someextent by substituent group R₃. Phenylboronic acid complexes of GeneralFormula CVI, wherein group R₃ is H, are stable in buffered aqueoussolutions over the approximate pH range 2.5 to 12.5. Phenylboronic acidcomplexes of General Formula CVI, wherein group R₃ is CH₃, are stable inbuffered aqueous solutions over the approximate pH range 3.5 to 12.5.Phenylboronic acid complexes of General Formula CVI, wherein group R₃includes an electronegative moiety, are stable in buffered aqueoussolutions over the approximate pH range less than 2.5 to 12.5.

The stability of the phenylboronic acid complex toward acid catalyzedhydrolysis is related to the pK_(a) of the hydroxamic acid participatingin the complex. The lower the pK_(a) of the hydroxamic acid moiety themore stable the complex. Consequently, phenylboronic acid complexes ofGeneral Formula CVI wherein group R₃ includes an electronegative moietyexhibit greater stability toward acid catalyzed hydrolysis than do thosein which R₃ is either H or CH₃.

It should be appreciated that the pH stability associated withbioconjugates of General Formula CVI, derived from4-aminomethyl-2,6-dihydroxybenzohydroxamic acid, is significantly betterthan that of bioconjugates of General Formula VI, derived from either 4-or 5-aminomethylsalicylhydroxamic acid, with respect to both acid andbase catalyzed hydrolysis.

The following examples present a detailed description of the synthesisof reagents of General Formula CI and General Formula CIII.

EXAMPLE CI Preparation of Methyl 4-Aminomethyl-2,6-dihydroxybenzoateHydrochloride ##STR40## Methyl 4-Methyl-2,6-diacetoxybenzoate

Methyl 4-methyl-2,6-dihydroxybenzoate (14.0 grams, 76.8 mmoles) wasdissolved in anhydrous dichloromethane (250 mL) and acetic anhydride (21mL, 223 mmoles) and anhydrous pyridine (18 mL, 223 mmoles) were addedcarefully. The solution was refluxed under dry nitrogen for 30 hours,then cooled to room temperature. The solution was washed twice with 1Maqueous hydrochloric acid (200 mL portions) and then with saturatedaqueous sodium chloride (200 mL). The dichloromethane solution was driedover anhydrous magnesium sulfate, filtered, and evaporated to anoff-white solid. This crude product was flash chromatographed on silicagel (250 grams) using hexanes:ethyl acetate (70:30 v/v!) as the elutingsolvent. Fractions containing the major product (R_(f) =0.3) were pooledand evaporated to dryness to afford 19.0 grams (98% yield) of a whitesolid of methyl 4-methyl-2,6-diacetoxybenzoate (m.p. 70°-71° C., opencapillary, uncorrected).

¹ H NMR (300 MHz, CHCl₃ -d) δ2.27 (singlet, 6H, COCH₃), 2.37 (singlet,3H, ArCH₃), 3.83 (singlet, 3H, OCH₃), 6.84 (singlet, 2H, ArH). ¹³ C NMR(75 MHz, CHCl₃ -d) δ20.9, 21.5, 52.4, 116.5, 122.1, 143.8, 150.2, 163.9,169.4.

Methyl 4-bromomethyl-2,6-diacetoxybenzoate

Methyl 4-methyl-2,6-diacetoxybenzoate (14.9 grams, 56.0 mmoles) wasdissolved in carbon tetrachloride (200 mL), and N-bromosuccinimide (11.1grams, 62.2 mmoles) and benzoyl peroxide (0.2 grams, 0.8 mmoles) wereadded. The mixture was refluxed under nitrogen. After 3.5 hours, anadditional portion (0.2 grams) of N-bromosuccinimide was added. Refluxwas continued for an additional hour. The reaction mixture was cooled toroom temperature and then in ice for 1 hour, and the solid removed byfiltration. The filtrate was evaporated to dryness. Hexanes (250 mL) wasadded to the residue, and the mixture was boiled until dissolution wasobtained. The hexanes solution was concentrated to about 75 mL when asolid just began to precipitate. The mixture was heated to dissolve thesolid, and the solution was allowed to cool slowly to room temperature.White crystals formed slowly, and the mixture was chilled in ice for 2hours to complete crystallization. The solid was filtered, washed withcold hexanes (100 mL), and dried in vacuo to afford 11.9 grams (62%yield) of methyl 4-bromomethyl-2,6-diacetoxy-benzoate (m.p. 93°-95° C.,open capillary, uncorrected). The product was contaminated with a smallamount of methyl 2,6-diacetoxy-4-dibromomethylbenzoate, which did notinterfere with subsequent reactions.

¹ H NMR (300 MHz, CHCl₃ -d) δ2.29 (singlet, 6H, COCH₃), 3.85 (singlet,3H, OCH₃), 4.41 (singlet, 2H, CH₂), 7.08 (singlet, 2H, ArH). ¹³ C NMR(75 MHz, CHCl₃ -d) δ20.9, 30.9, 52.7, 119.6, 121.8, 142.4, 150.2, 163.5,169.1.

Methyl 4-aminomethyl-2,6-dihydroxybenzoate hydrochloride

Methyl 4-bromomethyl-2,6-diacetoxybenzoate (45.8 grams, 133 mmoles) wasdissolved in N,N-dimethylformamide (150 mL), and sodium azide (8.8 g,135 mmoles) was added. The reaction mixture was stirred for 6 hours atroom temperature. Dichloromethane (350 mL) was added to the reactionmixture, and this solution was washed with water (250 ML), 1M aqueoushydrochloric acid (250 mL), and saturated aqueous sodium chloride (150mL). The organic solution was dried over anhydrous magnesium sulfate,filtered, and evaporated to a clear, pale yellow oil. The oil wasdissolved in methanol (750 mL) and transferred to a 2 L Parrhydrogenation flask. Palladium on carbon catalyst (10% w/w!, 1.5 g) inwater (10 mL) was added, followed by concentrated hydrochloric acid (15mL). The flask was affixed to a Parr hydrogenator, and the mixture wasshaken at room temperature under 30 psi of hydrogen for 24 hours. Thereaction mixture was filtered through a 0.45 mm nylon filter. Theretained solid was then washed with methanol (150 mL), and concentratedhydrochloric acid (7 mL) was added to the filtrate. The filtrate washeated to reflux for 2 hours, cooled to room temperature, and evaporatedto dryness to give an off-white solid. This solid was dissolved in hotdenatured ethanol (250 mL) and the solution was allowed to cool to roomtemperature. White crystals formed quickly. The mixture was then chilledfor 16-18 hours at 4° C. to complete crystallization. The solid wasfiltered, washed with a little cold ethanol (50 mL) and then diethylether (150 mL), and dried in vacuo over potassium hydroxide pellets toafford 16.0 grams (52% yield based on monobromo starting material) ofmethyl 4-aminomethyl-2,6-dihydroxybenzoate hydrochloride (m.p. >260° C.,open capillary, uncorrected).

¹ H NMR (300 MHz, DMSO-d₆) δ3.77 (singlet, 3H, OCH₃), 3.83 (singlet, 2H,CH₂), 6.44 (singlet, 2H, ArH), 8.35 (broad singlet, 3H, NH₃), 10.20(singlet, 2H, OH). ¹³ C NMR (75 MHz, DMSO-d₆) δ41.9, 52.1, 107.2, 107.3,138.5, 157.6, 168.3.

EXAMPLE CII Preparation of a Reagent of General Formula CI Methyl(6-Aminohexanoyl)aminomethyl-2,6-dihydroxybenzoate Hydrochloride##STR41## Methyl(N-tert-butoxycarbonyl-6-aminohexanoyl)aminomethyl-2,6-dihydroxybenzoate

Methyl 4-aminomethyl-2,6-dihydroxybenzoate hydrochloride (1.50 grams,6.4 mmoles) was suspended in anhydrous N,N-dimethylformamide (25 mL),and N,N-diisopropylethylamine (2.2 mL, 12.8 mmoles) was added, followedby N-tert-butoxycarbonyl-6-aminohexanoic acid succinimidyl ester (2.10grams, 6.4 mmoles). The mixture was stirred under dry nitrogen for 4hours, during which time all solids dissolved. The solvent was thenevaporated to leave a light brown syrup, which was partitioned betweenethyl acetate (70 mL) and 1M aqueous hydrochloric acid (50 mL). Thelayers were separated, and the ethyl acetate solution was washed withsaturated aqueous sodium bicarbonate (50 mL) and saturated aqueoussodium chloride (50 mL). The ethyl acetate solution was dried overanhydrous sodium sulfate, filtered, and evaporated to an amorphousoff-white solid. The solid was crystallized from ethyl acetate/hexanes,filtered, and dried in vacuo to afford 2.10 grams (80% yield) of methyl(N-tert-butoxycarbonyl-6-aminohexanoyl)aminomethyl-2,6-dihydroxybenzoate(m.p. 73°-76° C., open capillary, uncorrected).

¹ H NMR (300 MHz, DMSO-d₆) δ1.26 (multiplet, 2H, NHCH₂ CH₂ CH₂ CH₂ CH₂CO), 1.36 (multiplet, 2H, CH₂ CH₂ CO), 1.36 (singlet, 9H, C(CH₃)₃), 1.50(multiplet, 2H, NHCH₂ CH₂), 2.10 (triplet, J=7 Hz, 2H, CH₂ CH₂ CO), 2.87(quartet, J=6 Hz, 2H, NHCH₂ CH₂), 3.78 (singlet, 3H, OCH₃), 4.08(doublet, J=6 Hz, 2H, CH₂ Ar), 6.23 (singlet, 2H, ArH), 6.75 (triplet,J=6 Hz, 1H, CONHCH₂ CH₂), 8.26 (triplet, J=6 Hz, 1H, CONHCH₂ Ar), 10.04(singlet, 2H, OH). ¹³ C NMR (75 MHz, CHCl₃ -d) δ25.0, 26.0, 28.3, 29.3,35.3, 41.7, 52.0, 77.4, 104.3, 105.6, 145.8, 155.8, 158.2, 169.0, 172.4.

Methyl (6-aminohexanoyl)aminomethyl-2,6-dihydroxybenzoate hydrochloride

Methyl(N-tert-butoxycarbonyl-6-aminohexanoyl)aminomethyl-2,6-dihydroxybenzoate(2.00 grams, 4.87 mmoles) was dissolved in ethyl acetate (50 mL), anddry hydrogen chloride was bubbled slowly through the solution. Thereaction mixture warmed as the gas dissolved. After 5 minutes, the gaswas shut off, and the solution was stirred at room temperature. A whiteprecipitate formed in the solution. After 30 minutes, the reactionmixture was chilled in ice for 2 hours, then the solid was filtered,washed with ethyl acetate (50 mL) and then diethyl ether (50 mL), anddried in vacuo over potassium hydroxide pellets to afford 1.68 grams(99% yield) of methyl (6aminohexanoyl)aminomethyl-2,6-dihydroxybenzoatehydrochloride (m.p. shrinks at 145° C., decomposes with effervescence at152°-154° C., open capillary, uncorrected).

¹ H NMR (300 MHz, DMSO-d₆) δ1.29 (multiplet, 2H, H₃ CH₂ CH₂ CH₂ CH₂ CH₂CO), 1.55 (multiplet, 4H, NH₃ CH₂ CH₂ CH₂ CH₂ CH₂ CO), 2.13 (triplet,J=8 Hz, 2H, CH₂ CH₂ CO), 2.70 (multiplet, 2H, NH₃ CH₂ CH₂), 3.75(singlet, 3H, OCH₃), 4.07 (doublet, J=6 Hz, 2H, CH₂ Ar), 6.26 (singlet,2H, ArH), 8.05 (broad singlet, 3H, NH₃), 8.39 (triplet, J=6 Hz, 1H,CONHCH₂ Ar), 9.84 (broad singlet, 2H, OH). ¹³ C NMR(75 MHz, CHCl₃ -d)δ24.8, 25.6, 26.7, 35.1, 41.8, 52.0, 104.7, 105.5, 145.5, 158.1, 169.0,172.3.

EXAMPLE CIII Preparation of a Reagent of General Formula CI MethylN-Bromoacetyl-4-aminomethyl-2,6-dihydroxybenzoate ##STR42## MethylN-Bromoacetyl-4-aminomethyl-2,6-dihydroxybenzoate

Bromoacetic acid (0.30 grams, 2.16 mmol) was dissolved in dryN,N-dimethylformamide (15 mL) and N-hydroxysuccinimide (0.25 grams, 2.21mmol) and 1,3-dicyclohexylcarbodiimide (0.44 grams, 2.14 mmol) wereadded. The solution was stirred at room temperature for 1.25 hours.Methyl 4-aminomethyl-2,6-dihydroxybenzoate hydrochloride (0.50 grams,2.14 mmol) was added, followed by N,N-diisopropylethylamine (0.38 mL,2.15 mmol). The reaction mixture was stirred at room temperature for 2.5hours, and was then diluted with ethyl acetate (100 mL). The mixture wasfiltered, and the filtrate washed twice with 1N aqueous hydrochloricacid (100 mL then 50 mL) and with saturated aqueous sodium chloride (50mL). The organic solution was dried over anhydrous magnesium sulfate,filtered, and evaporated to dryness. The crude product was purified bychromatography on silica gel, eluting with hexanes:ethyl acetate (1:1v/v!). Fractions containing pure product were pooled and evaporated todryness to afford 0.43 grams of a white solid (63% yield) of methylN-bromoacetyl-4-aminomethyl-2,6-dihydroxybenzoate (m.p. 157°-158° C.).

¹ H NMR (300 MHz, DMSO-d₆) δ3.77 (singlet, 3H, OCH₃), 4.11 (singlet, 2H,BrCH₂), 4.13 (doublet, J=6 Hz, 2H, CH₂ NH), 6.25 (singlet, 2H, ArH),8.70 (triplet, J=6 Hz, 1H, CH₂ NH), 10.05 (singlet, 2H, OH). ¹³ C NMR(75 MHz, DMSO-d₆) δ42.3, 42.6, 52.1, 104.7, 105.7, 144.7, 158.2, 166.3,168.9.

EXAMPLE CIV Preparation of a Reagent of General Formula CI Methyl4-Glutarylaminomethyl-2,6-dihydroxybenzoate Succinimidyl Ester ##STR43##Methyl 4-glutarylaminomethyl-2,6-dihydroxybenzoate

Glutaric anhydride (2.3 grams, 20 mmoles) was dissolved in drytetrahydrofuran (100 mL), and the suspension was stirred under nitrogen.N,N-diisopropylethylamine (7.0 mL, 40 mmoles) was added, followed bymethyl 4-aminomethyl-2,6-dihydroxybenzoate hydrochloride (4.7 grams, 20mmoles). After stirring for 18 hours, the mixture was evaporated todryness, and the resulting solid was dissolved in dichloromethane (100mL) and allowed to sit at room temperature for 2 hours. Thedichloromethane solution was evaporated to dryness to give a thicksyrup, which was treated with cold 1M aqueous hydrochloric acid (100 mL)for 30 minutes in ice. An off-white solid precipitated, which wasfiltered, washed with cold water, and dried in vacuo to afford 5.5 grams(88% yield) of methyl 4-glutarylaminomethyl-2,6-dihydroxybenzoate (m.p.163°-164° C.).

¹ H (300 MHz, DMSO-d₆) δ1.74 (quintet, J=8 Hz, 2H, CH₂ CH₂ CH₂), 2.16(triplet, J=8 Hz, 2H, CH₂ CONH), 2.20 (triplet, J=8 Hz, 2H, HO₂ CCH₂),3.78 (singlet, 3H, OCH₃), 4.10 (doublet, J=6 Hz, 2H, ArCH₂ NH), 6.23(singlet, 2H, ArH), 8.31 (triplet, J=6 Hz, 1H, NH), 10.05 (broadsinglet, 3H, OH and CO₂ H). ¹³ C NMR (75 MHz, DMSO-d₆) δ20.7, 33.1,34.4, 41.8, 52.1, 104.4, 105.6, 145.7, 158.2, 169.0, 172.0, 174.5.

Methyl 4-glutarylaminomethyl-2,6-dihydroxybenzoate succinimidyl ester

Methyl 4-glutarylaminomethyl-2,6-dihydroxybenzoate (2.0 grams, 6.4mmoles) was dissolved in dry tetrahydrofuran (80 mL), andN-hydroxysuccinimide (0.8 grams, 7.0 mmoles) and1,3-dicyclohexylcarbodiimide (1.3 grams, 6.4 mmoles) were added. Themixture was stirred at room temperature under dry nitrogen, and thesolids rapidly dissolved. After about 20 minutes, a white precipitateformed. The reaction mixture was stirred 16-18 hours, then chilledseveral hours at -20° C. The mixture was filtered cold, and the solidwashed with a little tetrahydrofuran (25 mL). The combined filtrateswere evaporated to dryness, and the residue was crushed under ice-coldwater (25 mL). The solid was filtered, washed quickly with cold water(25 mL) and then diethyl ether (100 mL), and dried in vacuo. The solidwas recrystallized from ethyl acetate/hexanes to afford 2.5 grams (95%yield) of methyl 4-glutarylaminomethyl-2,6-dihydroxybenzoatesuccinimidyl ester (m.p. 161-°163° C.).

¹ H NMR (300 MHz, DMSO-d₆) δ1.83 (quintet, J=8 Hz, 2H, CH₂ CH₂ CH₂),2.26 (triplet, J=8 Hz, 2H, CH₂ CONH), 2.73 (triplet, J=8 Hz, 2H, NO₂CCH₂), 2.80 (singlet, 4H, COCH₂ CH₂ CO), 3.77 (singlet, 3H, OCH₃), 4.10(doublet, J=6 Hz, 2H, ArCH₂ NH), 6.24 (singlet, 2H, ArH), 8.35 (triplet,J=6 Hz, 1H, NH), 10.04 (broad singlet, 2H, OH). ¹³ C NMR (75 MHz,DMSO-d₆) δ20.4, 25.4, 29.7, 33.6, 41.8, 52.0, 104.5, 105.6, 145.5,158.1, 168.9, 170.5, 171.4.

EXAMPLE CV Preparation of a Reagent of General Formula CI Methyl4-Glutarylaminomethyl-2,6-dihydroxybenzoic Acid Hydrazide ##STR44##Methyl 4-glutarylaminomethyl-2,6-dihydroxybenzoateN-tert-butyloxycarbonylhydrazide

Methyl 4-glutarylaminomethyl-2,6-dihydroxybenzoate succinimidyl ester(1.6 grams, 3.9 mmoles) was suspended in dry dichloromethane (50 mL),and tert-butylcarbazate (0.50 grams, 3.9 mmoles) was added. The reactionwas stirred at room temperature for 24 hours. The pale solution was thenwashed twice with saturated aqueous sodium bicarbonate (50 mL portions),0.1M aqueous hydrochloric acid (50 mL), and saturated aqueous sodiumchloride (50 mL). It was then dried over anhydrous sodium sulfate,filtered, and evaporated to dryness. The residue was flashchromatographed on a 2.5×150 cm column of silica gel, eluting with ethylacetate. Fractions containing pure product were pooled and evaporated toa clear syrup. This syrup was crystallized from ethyl acetate:hexanes at-20° C. The solid was filtered, washed with cold hexanes, and dried invacuo to afford 1.0 grams (62% yield) of methyl4-Glutarylaminomethyl-2,6-dihydroxybenzoateN-tert-butyloxycarbonylhydrazide (m.p. 95°-100° C., softens at 88° C.,open capillary, uncorrected).

¹ H (300 MHz, DMSO-d₆) δ1.37 (singlet, 9H, (CH₃)₃ C), 1.74 (quintet, J=7Hz, 2H, CH₂ CH₂ CH₂), 2.07 (triplet, J=7 Hz, 2H, CH₂ CONHCH₂), 2.15(triplet, J=7 Hz, 2H, HNHNOCCH₂), 3.77 (singlet, 3H, OCH₃), 4.09(doublet, J=6 Hz, 2H, ArCH₂ NH), 6.23 (singlet, 2H, ArH), 8.28 (triplet,J=6 Hz, 1H, NH), 8.65 (singlet, 1H, NHNHCOCH₂), 9.47 (singlet, OCONHNH),9.96 (singlet, 2H, OH). ¹³ C NMR (75 MHz, DMSO-d₆) δ21.2, 28.0, 32.7,34.7, 41.7, 52.1, 79.1, 104.4, 105.6, 145.7, 155.5, 158.2, 169.0, 171.7,172.0.

Methyl 4-Glutarylaminomethyl-2,6-dihydroxybenzoic Acid Hydrazide

Methyl 4-glutarylaminomethyl-2,6-dihydroxybenzoateN-tert-butyloxycarbonyl hydrazide (0.50 grams, 1.2 mmoles) is dissolvedin dry tetrahydrofuran (25 mL), and dry hydrogen chloride was bubbledslowly through the solution. The reaction mixture warmed as the gasdissolved. After 5 minutes, the gas was shut off, and the solution wasstirred at room temperature. A white precipitate formed in the solution.After 30 minutes, the reaction mixture was chilled in ice for 2 hours,then the solid was filtered, washed with diethyl ether (50 mL), anddried in vacuo over potassium hydroxide pellets to afford 0.42 grams(98% yield) of methyl 4-glutarylaminomethyl-2,6-dihydroxybenzoic acidhydrazide (m.p. 158°-159° C., decomposed with effervescence, opencapillary, uncorrected).

¹ H (300 MHz, DMSO-d₆) δ1.76 (quintet, J=7 Hz, 2H, CH₂ CH₂ CH₂), 2.16(triplet, J=7 Hz, 2H, CH₂ CONHCH₂), 2.21 (triplet, J=7 Hz, 2H, H₃NHNOCCH₂), 3.75 (singlet, 3H, OCH₃), 4.07 (doublet, J=6 Hz, 2H, ArCH₂NH), 5.00-7.00 (very broad singlet, 2H, OH), 6.25 (singlet, 2H, ArH),8.39 (triplet, J=6 Hz, 1H, NH), 10.49 (broad singlet, 3H, NHNH₃), 11.09(singlet, 1H, NHNH₃). ¹³ C NMR(75 MHz, CHCl₃ -d) δ20.9, 32.2, 34.4,41.8, 52.0, 104.8, 105.6, 145.5, 158.1, 169.0, 171.6, 171.9.

EXAMPLE VI Preparation of a Reagent of General Formula CIII Synthesis of4-Glutarylaminomethyl-2,6-dihydroxybenzohydroxamic Acid Hydrazide##STR45## 4-Glutarylaminomethyl-2,6-dihydroxybenzohydroxamic AcidN-tert-Butyloxycarbonylhydrazide

Methyl 4-glutarylaminomethyl-2,6-dihydroxybenzoateN-tert-butyloxycarbonylhydrazide (0.30 grams, 0.7 mmoles) was added to acooled (ice/water bath) solution of hydroxylamine sulfate (0.12 grams,0.7 mmoles), sodium hydroxide (0.15 grams, 3.6 mmoles) and sodiumsulfite (0.05 grams, 0.4 mmoles) in water (6 mL). The suspension wasstirred for 24 hours in the dark, allowing it to warm to roomtemperature. The solution was dilute with 1M hydrochloric acid (4 mL),and extracted twice with ethyl acetate (15 mL portions). The combinedethyl acetate extracts were washed with water (10 mL) and saturatedaqueous sodium chloride (10 mL), dried over anhydrous magnesium sulfate,filtered, and evaporated to dryness. The residue was dissolved in aminimum volume of ethyl acetate, and diethyl ether was added toprecipitate an orange gummy material. The gum was triturated under drydiethyl ether to produce a solid, which was filtered, washed with ether,and dried in vacuo to afford 0.21 grams (69% yield) of4-glutarylaminomethyl-2,6-dihydroxybenzohydroxamic acidN-tert-butyloxycarbonylhydrazide (m.p. decomposed with effervescence at60°-65° C., open capillary, uncorrected).

¹ H (300 MHz, DMSO-d₆) δ1.37 (singlet, 9H, (CH₃)₃ C), 1.73 (quintet, J=7Hz, 2H, CH₂ CH₂ CH₂), 2.07 (triplet, J=7 Hz, 2H, CH₂ CONHCH₂), 2.15(triplet, J=7 Hz, 2H, HNHNOCCH₂), 4.08 (doublet, J=6 Hz, 2H, ArCH₂ NH),6.22 (singlet, 2H, ArH), 8.28 (triplet, J=6 Hz, 1H, NH), 8.65 (singlet,1H, NHNHCOCH₂), 9.47 (singlet, OCONHNH), 10.77 (broad singlet, 1H,NHOH), 12.30 (broad singlet, 3H, ArOH and NHOH). ¹³ C NMR (75 MHz,DMSO-d₆) δ21.3, 28.1, 32.7, 34.7, 41.7, 79.1, 100.1, 105.6, 145.8,155.6, 159.8, 167.5, 171.7, 172.0.

4-Glutarylaminomethyl-2,6-dihydroxybenzohydroxamic acid hydrazidetrifluoroacetate

4-Glutarylaminomethyl-2,6-dihydroxybenzohydroxamic acid tert-butylcarbazate (0.09 grams, 0.2 mmoles) was suspended in dry dichloromethane(5 mL), and trifluoroacetic acid (0.5 mL) was added. The solid dissolvedon addition of the acid, and the reaction was stirred at roomtemperature for three hours. After one hour, an orange precipitate hadformed. The precipitate was collected by filtration and dried in vacuoover potassium hydroxide pellets to afford 0.10 grams (100% yield) of4glutarylaminomethyl-2,6-dihydroxybenzohydroxamic acid hydrazidetrifluoroacetate (m.p. 104°-108° C., open capillary, uncorrected).

¹ H (300 MHz, DMSO-d₆) δ1.80 (quintet, J=7 Hz, 2H, CH₂ CH₂ CH₂), 2.17(triplet, J=7 Hz, 2H, CH₂ CONHCH₂), 2.23 (triplet, J=7 Hz, 2H,HNHNOCCH₂), 4.09 (doublet, J=6 Hz, 2H, ArCH₂ NH), 6.22 (singlet, 2H,ArH), 8.32 (triplet, J=6 Hz, 1H, NH), 8.95-10.60 (very broad singlet,5H, NHNH₃ and NHOH), 11.80-12.95 (very broad singlet, 3H, ArOH andNHOH). ¹³ C NMR (75 MHz, DMSO-d₆) δ20.9, 32.2, 34.4, 41.8, 100.1, 105.7,145.8, 158.5, 159.0, 159.9, 167.5, 171.8, 171.9.

EXAMPLE CVII Preparation of Conjugates of General Formula CIV

Synthesis of 5'-PBA-labeled Oligodeoxyribonucleotide Conjugates

Oligodeoxyribonucleotide 7172 (sequence 5'-GATTACGCCAGTTGTACGGAC-3') wassynthesized on a 1 μmole scale using standard automated phosphoramiditechemistry (Beckman Instruments Oligo 1000 and associated reagents). Aprotected amine-containing phosphor-amidite (Aminolink 2, AppliedBiosystems or UniLink Amino Modifier, Clontech) was employed on the sameinstrument to introduce one to four, reactive primary amines onto the5'-end of the oligodeoxyribonucleotide using standard chemistry. Thecompleted oligodeoxyribonucleotide was then cleaved from the support andthe nucleobases deprotected using an UltraFast Deprotection kit (BeckmanInstruments) and the protocol supplied by the manufacturer.

The amino-oligonucleotides were purified by ethanol precipitation,dissolved in 0.8 mL of 0.1M NaHCO₃, and condensed with phenylboronicacid reagent (PBA-Z-NHS) having a reactive N-hydroxysuccinimidyl estermoiety (5 mgs per mmole of primary amino groups on theamino-oligonucleotide in 0.2 mL of anhydrous N,N-dimethylformamide) for2-18 hours at room temperature.

The crude PBA-modified oligonucleotide was isolated from the reactionmixture by gel filtration on a KwikSep Dextran column (Pierce Chemical)in 0.1M aqueous triethylammonium acetate, pH 6.5. The PBA-modifiedoligonucleotide was then concentrated in a vacuum centrifuge to 1 mL,and purified by reverse phase HPLC on a 4.6 mm×250 mm C18 column, with atriethylammonium acetate-acetonitrile gradient. The desired product peakwas collected and evaporated to a small pellet in a vacuum centrifuge,dissolved in 0.5 mL of water, and stored frozen.

Preparation of Salicylhydroxamic Acid Magnetic Beads

Ten milliliters of unmodified M280 or M450 magnetic beads (Dynal) weregradually dehydrated into acetonitrile, and converted to aldehydemodified beads using oxalyl chloride activated N,N-dimethylsulfoxide andtriethylamine in dichloromethane at -78° C. The resulting aldehydebearing beads were gradually rehydrated and suspended in 5 mL of 0.1Msodium acetate pH 5.5. The aldehyde groups were coupled with4-glutarylaminomethylsalicylhydroxamic acid hydrazide (SHA--Z--NHNH₂) byadding 15-25 mgs dissolved in 200 uL N,N-dimethylsulfoxide, and rotatingcoupling reaction over night at room temperature. The beads were thenwashed extensively with water and stored in 5 mL of 10% ethanol.

Preparation of Salicylhydroxamic Acid (SHA) Sepharose 4B

SHA-Sepharose 4B was prepared by mixing 130 mg of(6-aminohexanoyl)-4-aminomethylsalicylhydroxamic acid (SHA--Z--NH₂),dissolved in 30 mL 0.2M NaHCO₃, with. 6.5 g HCl washed CNBr activatedSepharose 4B (Pharmacia) overnight at room temperature. After thecoupling reaction, 2 mL of 0.5M Tris, pH 8.5 were added and the gelslurry mixed at room temperature for 1 hour, and washed with water, 0.5MNaCl, and water again. The resulting SHA-Sepharose 4B was suspended in30 mL of 20% ethanol, and stored at 4° C.

Preparation of 2,6-Dihydroxybenzohydroxamic Acid (DHBHA) Sepharose 4B

DHBHA-Sepharose 4B was prepared by mixing 114 mg methyl(6-amino-hexanoyl)-4-aminomethyl-2,6-dihydroxybenzoate DHBA(OMe)--X--NH₂!, dissolved in 30 mL 0.2M NaHCO₃, with 5 g HCl washed CNBr activatedSepharose 4B (Pharmacia), overnight at room temperature. After thecoupling reaction, the gel was washed with water and suspended in 50 mL0.1M NH₂ OH, pH 9, and rotated at room temperature for two hours.Finally, the gel was washed with water and suspended in 30 mL of 20%ethanol, and stored at 4° C.

Preparation of a Phenylboronic Acid-α-Biotin Antibody Conjugate

One milliliter of α-Biotin monoclonal IgG₁ antibody (6.5 mg/mL in 0.1MNaHCO₃) was conjugated with 440 nmoles of PBA--Z--NHS (7.4 ul of 60 mMPBA--Z--NHS dissolved in N,N-dimethylsulfoxide) for 1 hour at roomtemperature. Unconjugated PBA--Z--NHS and its hydrolysis products wereremoved by dialysis. The ultra-violet absorbance spectrum of theresulting conjugate (PBA-α-Biotin) exhibited an increase in A₂₆₀relative to A₂₈₀ consistent with phenylboronic acid modification.

Preparation of a Phenylboronic Acid-Alkaline Phosphatase Conjugate

One milliliter of alkaline phosphatase (Sigma, 6 mg/mL) was dialyzedagainst one liter of 0.1M NaHCO₃, and conjugated with 700 nmoles ofPBA--Z--NHS (10 uL of 70 mM stock in N,N-dimethylformamide) for twohours on ice. Unconjugated PBA--Z--NHS and its hydrolysis products wereremoved by dialysis in 0.1M NaHCO₃. The ultra-violet absorbance spectrumof the resulting conjugate (PBA-AP) exhibited an increase in A₂₆₀relative to A₂₈₀ consistent with phenylboronic acid modification. Theconjugate was stored at 4° C.

Preparation of a 2,6-Dihydroxybenzohydroxamic Acid-Alkaline PhosphataseConjugate

One milliliter of alkaline phosphatase (Sigma, 6 mg/mL) was dialyzedagainst one liter of 0.1M NaHCO₃, and conjugated with 714 nmoles ofmethyl 4-glutarylaminomethyl-2,6-di-hydroxybenzoate succinimidyl esterDHBA(OMe)--Z--NHS! (10.5 uL of 68 mM in N,N-dimethylformamide) for twohours on ice. The methyl ester of the conjugate was converted to ahydroxamic acid by adding one milliliter of 2M NH₂ OH, pH 10, andincubating the reaction at 4° C. for six days. The NH₂ OH reactionmixture was then dialyzed against 0.1M NaHCO₃ and stored at 4° C.

Preparation of a Salicylhydroxamic Acid-Goat-α-Mouse Antibody Conjugate

Two milliliters of goat α-mouse antibody (Rockland, 8.8 mg/mL in 0.1MNaHCO₃) were conjugated with 2.35 umoles of methyl4-glutarylaminomethylsalicylate succinimidyl ester SA(OMe)--Z--NHS! for1 hour at room temperature. The methyl ester of the conjugate wasconverted to a hydroxamic acid by adding two milliliters of 2M NH₂ OH,pH 10, adjusting the pH to 10 with 1N NaOH, and incubating the reactionat room temperature for three days. NH₂ OH and unconjugatedSA(OMe)--X--NHS and its hydrolysis products were removed by gelfiltration on a G-25 Sephadex column (Pharmacia) in 0.1M NaHCO₃, and theconjugate (SHA-goat α-mouse) was stored at 4° C.

Preparation of a 2,6-Dihydroxybenzohydroxamic Acid-Goat α-Mouse Antibody

Two milliliters of goat α-mouse antibody (Rockland, 8.8 mg/mL in 0.1MNaHCO₃) were conjugated with 2.35 umoles of with DHBA(OMe)--Z--NHS for 1hour at room temperature. The methyl ester of the conjugate wasconverted to a hydroxamic acid by adding two milliliters of 2M NH₂ OH,pH 10, adjusting the pH to 10 with 1N NaOH, and incubating the reactionat room temperature for three days. NH₂ OH and unconjugatedDHBA(OMe)--X--NHS and its hydrolysis products were removed by gelfiltration on a G-25 Sephadex column (Pharmacia) in 0.1M NaHCO₃, and theconjugate (DHBHA-goat α-mouse) stored at 4° C.

Polymerase Chain Reaction (PCR) Protocol

A region of Lambda DNA (801 bp) was amplified by the polymerase chainreaction. The PCR reaction contained 200 uM dATP, dCTP, dGTP, and dTTPin addition to Biotin- and PBA- modified oligonucleotide primers at 1 uMin 1×PCR Buffer II (Perkin Elmer), Lambda DNA (1 ng/uL), and 1 U ofThermus aquaticus DNA polymerase. The reaction mixture was denatured at92° C. for one minute and amplified by 35 cycles of PCR at 95° C. for 10seconds, 62° C. for 20 seconds, and 72° C. for 30 seconds, with a finalextension at 72° C. for 5 minutes. The reaction produced 50-100 ng ofamplified product (801 bp), which exhibited retarded mobility relativeto unmodified PCR product during electrophoresis on 1% agarose gels in50 mM Tris, 100 mM borate, 2 mM EDTA, pH 8.3.

EXAMPLE CVIII Preparation of Bioconjugates of General Formula CVISandwich Hybridization Detection of Nucleic Acid Probes

A 42-mer oligonucleotide was hybridized with two 21-mer oligonucleotidesbearing 5'-PBA and Biotin labels in 1.5M NaCl, 150 mM sodium citrate, pH7, at 45° C. for ten minutes. Twenty-five microliters of thehybridization mixture was mixed with twenty five microliters of M280streptavidin-magnetic particles (Dynal) in a polypropylene microtiterplate well. After 30 minutes, the magnetic particles were captured inthe bottom of the well with a magnetic plate, and washed five times with150 mM NaCl, 20 mM Tris-HCl, 0.02% Tween20, pH 8.

One hundred microliters of DHBHA-AP (1 ug/mL in 1 mg/mL BSA, 140 mMNaCl, 10 mM Tris-HCl, pH 8) were added to the magnetic particles andmixed well. After 30 minutes, the magnetic particles were captured inthe bottom of the well with a magnetic plate, and washed six times with150 mM NaCl, 20 mM Tris-HCl, 0.02% Tween20, pH 8. Alkaline phosphatasesubstrate (1 mg/mL p-nitrophenylphosphate in 1M diethanolamine buffer, 1mM MgCl₂, 0.1 mM ZnCl₂, pH 10.4) was added, and incubated at 37° C. for90 minutes. The Absorbance at 405 nm (A₄₀₅) was measured with an ELISAplate reader (Molecular Devices).

A strong A₄₀₅ was produced when all components of the hybridizationsandwich were present, and the signal was proportional to the amount of42-mer present. Experimental controls lacking either the 42-mer, theBiotin-oligonucleotides and PBA-oligonucleotides did not produce asignificant A₄₀₅.

EXAMPLE CIX Preparation of Bioconjugates of General Formula CVI

Detection of PBA-Labeled PCR Product on DHBHA-Coated Microtiter Plates

The wells of a polystyrene microtiter plate (Becton Dickinson) werecoated with DHBHA by filling the wells with 200 uL of DHBHA-goat α-mouseconjugate (30 ug/mL in 0.1M NaHCO₃ pH 9.0) and incubating overnight at4° C. The coating solution was removed and the plate backcoated with 5mg/mL BSA (300 ul per well in 0.2M NaHCO₃, pH 9.0) for 1 hour at roomtemperature. The BSA solution was removed by washing the plate fivetimes with ELISA Wash Buffer (150 mM NaCl, 20 mM Tris-HCl, 0.02% Tween20, pH 8.0).

One hundred microliters of unpurified PBA and biotin labeled PCR productwere added to 900 ul of 1.5N NaCl, 150 mM sodium citrate, pH 7.0(10×SSC) and serially-diluted in 10×SSC. One hundred microliters of thediluted PCR products were added to the wells and incubated for one hourat room temperature. The plate was then washed five times with ELISAWash Buffer, and 100 ul of Streptavidin-Alkaline Phosphatase (BoehringerMannheim, 0.2 U/mL in 1 mg/ml BSA, 140 mM NaCl, 10 mM Tris-HCl, pH 8.0)were added to each well and incubated for thirty minutes at roomtemperature.

The plate was washed 5 times with ELISA Wash Buffer, and 200 ul ofp-nitrophenylphosphate (1 mg/mL in diethanolamine, 1 mM MgCl₂, 0.1 mMZnCl₂, pH 10.4) were added to the plate and incubated at 37° C. for30-60 minutes. Less than 1 uL of PCR product was detected. PCR productlacking either PBA or biotin labels was not detected.

EXAMPLE CX Preparation of Bioconjugates of General Formula CVI Detectionof PBA-Labeled PCR Product

Detection of PBA-Labeled PCR Product on SHA-Magnetic Beads

PBA-labeled PCR product (0.02 μL-5 μL) was diluted into 25-100 μL of1.5M NaCl, 150 mM sodium citrate, pH 7 (10×SSC), and added to apolypropylene microtiter plate well containing SHA-magnetic particles(10-50 ul). The particles and PCR product were mixed occasionally for30-60 minutes at room temperature. The magnetic particles were capturedin the bottom of the wells with a magnetic plate and washed five timesin 150 mM NaCl, 20 mM Tris-HCl, 0.02% Tween 20, pH 8 (ELISA WashBuffer). One hundred microliters of streptavidin alkaline-phosphatase(Boehringher Mannheim, 0.2 U/mL in 1 mg/mL BSA, NaCl, Tris-HCl, pH 8)were added and mixed with the magnetic particles for 30 minutes at roomtemperature. The magnetic particles were captured in the bottom of thewells with a magnetic plate and washed 5 times with ELISA Wash. Alkalinephosphatase substrate was added (1 mg/ml p-nitrophenyl phosphate in 1Mdiethanolamine buffer, 1 mM MgCl₂, 0.1 mM ZnCl₂, pH 10.4), and the colordeveloped at 37° C. for 10-60 minutes. The lower limit of detection was50 pg of PCR product.

EXAMPLE CXI Preparation of Bioconjugates of General Formula CVIDetection of a PBA-Labeled Oligonucleotide Hybrid

Detection of a PBA-Labeled Oligonucleotide Hybridized to aBiotin-Labeled Oligonucleotide

A 42-mer oligonucleotide was hybridized with two 21-mer oligonucleotidesbearing 5'-PBA and Biotin labels in 1.5M NaCl, 150 mM sodium citrate, pH7, at 45 C. for ten minutes. Twenty-five microliters of thehybridization mixture was mixed with 1-50 uL of SHA-magnetic particles(Dynal, M450) in a polypropylene microtiter plate well. After 30minutes, the magnetic particles were captured in the bottom of the wellwith a magnetic plate, and washed five times with 150 mM NaCl, 20 mMTris-HCl, 0.02% Tween 20, pH 8.

One hundred microliters of SHA-AP (1 ug/mL in 1 mg/mL BSA, 140 mM NaCl,10 mM Tris-HCl, pH 8) were added to the magnetic particles and mixedwell. After 30 minutes, the magnetic particles were captured in thebottom of the well with a magnetic plate, and washed six times with 150mM NaCl, 20 mM Tris-HCl, 0.02% Tween 20, pH 8. The particles were mixedwith alkaline phosphatase substrate (1 mg/mL p-nitrophenyl phosphate in1M diethanolamine buffer, 1mM MgCl₂, 0.1 mM ZnCl₂, pH 10.4) andincubated at 37° C. for 90 minutes. The A₄₀₅ was measured with a ELISAplate reader (Molecular Devices). As little as 45 pg of oligonucleotide42-mer was detected. Experimental controls lacking either the 42-mer, orthe PBA or Biotin labeled oligonucleotides did not produce a significantA₄₀₅.

EXAMPLE CXII Preparation of Bioconjugates of General Formula CVI

Immobilization of a PBA-α-Biotin Conjugate on SHA-Sepharose 4B

One mg of PBA-α-Biotin, diluted to 1 mL with Tris buffered saline, wasapplied to small column of SHA-Sepharose 4B (1.0×2.0 cm), and washedextensively with Tris buffered saline. The size of the A₂₈₀ peak of thematerial not binding to the column indicated that almost all of thePBA-conjugate was immobilized on the column.

Biotin binding activity of the column was assayed by applying to thecolumn 5 mL of 1 ug/mL biotinylated alkaline phosphatase in Trisbuffered saline containing 5 mg/mL bovine serum albumin (BSA). A sampleof the peak of the material flowing through the column was collected forcomparison of the enzymatic activity with a sample of the alkalinephosphatase dilution applied to column. After applying the sample, thecolumn was washed with 20 mL of buffer. After washing, a very smallsample of column material (25 uL liquid containing about 1 uL gel) wascollected to measure the enzymatic activity bound to the gel as a resultof capture by the immobilized α-biotin antibody.

The alkaline phosphatase activity was measured by incubating 25 uL ofthe enzyme samples in 250 uL of 1 mg/mL p-nitrophenylphosphate in 1Mdiethanolamine buffer, 1 mM MgCl₂, and 0.1 mM ZnCl₂, pH 10.4, for 20minutes and then adding 650 uL of 0.1M NaHCO₃, 10 mM EDTA. Relative to abuffer blank, the A₄₀₅ of the sample applied to the column was 1.57,while the A₄₀₅ of the peak of the material not retained by the columnwas only 0.042, indicating that virtually all the enzyme conjugate wascaptured by the column. The small amount of gel assayed produced an A₄₀₅of 1.30, demonstrating that the enzyme was in fact captured by thecolumn.

EXAMPLE CXIII Preparation of Bioconjugates of General Formula CVI

Immobilization of a PBA-Alkaline Phosphatase Conjugate on SHA-MagneticBeads

PBA-conjugated alkaline phosphatase was diluted to 5 ug/mL in Trisbuffered saline containing 5 mg/mL bovine serum albumin. Two hundredmicroliters of diluted PBA-conjugated enzyme were mixed with 5, 10, or20 uL of SHA-magnetic beads (Dynal, M280). The enzyme was also mixedwith 40 uL of unmodified beads as a control. The beads were mixed gentlyfor 10 minutes on ice, after which the beads were captured with a rareearth magnet and washed 4 times with Tris buffered saline. The beadswere then suspended in 250 uL of 1 mg/mL p-nitrophenylphosphate in 1Mdiethanolamine buffer, 1 mM MgCl₂, and 0.1 mM ZnCl₂, pH 10.4, and mixedoccasionally at 37° C. for 10 minutes. The reactions were terminatedwith 750 uL of Tris buffered saline, 5 mM EDTA. The A₄₀₅ relative to abuffer blank was measured to determine the alkaline phosphatase activitybound to the magnetic beads. The control beads produced an A₄₀₅ of only0.15, while the SHA-magnetic beads produced an A₄₀₅ of 0.62, 0.97, and1.33 for 5, 10, and 20 uL of beads, respectively, indicating theimmobilization of significant amounts of PBA-AP conjugate on the surfaceof the beads.

We claim:
 1. A conjugate of a biologically active species with a reagenthaving the general formula of General Formula CII: ##STR46## whereingroup Z comprises a spacer selected from a saturated or unsaturatedchain of up to about 6 carbon equivalents in length, an unbranchedsaturated or unsaturated chain of from about 6 to 18 carbon equivalentsin length with at least one of intermediate amide and disulfidemoieties, and a polyethylene glycol chain of from about 3 to 12 carbonequivalents in length;wherein group R₂ is one of an alkyl and amethylene with an electronegative moiety; and wherein BAS is abiologically active species.
 2. The conjugate of claim 1, wherein groupR₂ is selected from one of CH₃, CH₂ CH₃, CH₂ CN, CH₂ COOH, CH₂ CONH₂,and CH₂ OCH₃.
 3. The conjugate of claim 1, wherein group Z is anunbranched alkyl chain of general formula (CH₂)_(n), wherein n=1 to 6.4. The conjugate of claim 1, having the general formula: ##STR47##wherein group R₂ is CH₃.